Use of cationic lipids to deliver CAS9

ABSTRACT

Compositions, methods, strategies, kits, and systems for the supercharged protein-mediated delivery of functional effector proteins into cells in vivo, ex vivo, or in vitro are provided. Compositions, methods, strategies, kits, and systems for delivery of functional effector proteins using cationic lipids and cationic polymers are also provided. Functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins; TALE nuclease, and zinc finger nucleases), deaminases, and other gene modifying/editing enzymes. Functional effector proteins include TALE effector proteins, e.g., TALE transcriptional activators and repressors, as well as TALE nucleases. Compositions, methods, strategies, and systems for the delivery of functional effector proteins into cells is useful for therapeutic and research purposes, including, but not limited to, the targeted manipulation of a gene associated with disease, the modulation of the expression level of a gene associated with disease, and the programming of cell fate.

RELATED APPLICATION

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application Ser. No. 61/874,746, filed Sep. 6, 2013,which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with U.S. Government support under GM095501awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Macromolecular delivery into mammalian cells is an attractive approachfor cell manipulation, as it would allow modulation of gene expressionand modification of the genome, which, in turn, would open new avenuesfor research and enable the therapeutic targeting of molecules currentlyviewed as “undruggable” by small molecules. In particular, recombinantnucleases targeting genes or alleles associated with disease have greatpotential as therapeutic agents. The current methods of macromoleculardelivery include viral delivery of nucleic acid molecules,receptor-mediated delivery of nucleic acids or proteins, and the use ofprotein fusions with cell-penetrating peptides such as TAT, Arg9, orPenetratin for the delivery of proteins. Each of these delivery systemsoffers benefits for particular applications; in most cases, however,questions regarding efficacy, cytotoxicity, and ease of preparationremain. Easily prepared reagents capable of effectively deliveringmacromolecules (e.g., functional effector proteins) to a variety of celllines without significant cytotoxicity or other adverse side effectremain of considerable concern.

Most proteins do not spontaneously enter mammalian cells and are thusnaturally limited in their use as research tools and their potential astherapeutic agents. Techniques for the delivery of proteins intomammalian cells have been developed recently to address intracellulartargets. These techniques include the use of lipid-based reagents(Zelphati et al., J. Biol. Chem. 276, 35103-35110, 2001), nanoparticles(Hasadsri et al., J. Biol. Chem., 2009), vault ribonucleoproteinparticles (Lai et al., ACS Nano 3, 691-699, 2009); genetic or chemicalfusion to receptor ligands (Gabel et al., J. Cell Biol. 103, 1817-1827,1986; Rizk et al., Proc. Natl. Acad. Sci. U.S.A. 106, 11011-11015,2009); and fusion to cell-penetrating peptides (Wadia et al., Curr.Protein Pept. Sci. 4, 97-104, 2003; Zhou et al., Cell Stem Cell 4,381-384, 2009). Perhaps the most common method for protein delivery isgenetic fusion to protein transduction domains (PTDs) including theHIV-1 transactivator of transcription (Tat) peptide and polyargininepeptides. These cationic PTDs promote association with negativelycharged cell-surface structures and subsequent endocytosis of exogenousproteins. Both Tat and polyarginine have been used to deliver a varietyof macromolecules into cells both in vitro and in vivo (Wadia et al.,Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al., Cell Stem Cell 4,381-384, 2009; Myou et al., J. Immunol. 169, 2670-2676, 2002; Bae etal., Clin. Exp. Immunol. 157, 128-138, 2009; Schwarze et al., Science285, 1569-1572, 1999). Despite these advances, intracellular targetsremain difficult to affect using exogenous proteins, and even modestsuccess can require toxic concentrations of the respective transductionagent due to the low efficiency with which proteins are functionallydelivered into cells (Zhou et al., Cell Stem Cell 4, 381-384, 2009; Wanget al., Nat. Biotechnol. 26, 901-908, 2008). Therefore, there remains aneed for better delivery systems for getting functional effectorproteins into cells to target intracellular biomolecules.

SUMMARY OF THE INVENTION

The present disclosure provides novel systems, compositions,preparations, kits, and related methods for delivering functionaleffector proteins, such as, for example, site-specific proteins thatbind nucleic acids, into cells using a supercharged protein (e.g., apositively charged supercharged protein), a cationic polymer, or acationic lipid. In some embodiments, the nucleases are TALE nucleases,RNA-programmable nucleases or engineered RNA-programmable genome-editingenzymes (such as Cas9 and variants or fusions thereof), or zinc fingernucleases. In some embodiments, the transcription factors are TALEtranscriptional activators or repressors. In some embodiments, theeffector proteins are recombinases. As described in greater detailherein, fusing or associating functional effector proteins (e.g.,nucleases, transcriptional activators/repressors, Cas9 proteinsincluding variants and fusions thereof, etc.) with positively chargedsupercharged proteins allows for delivery of the proteins to theinterior of cells, for example to affect gene expression or genomicmodifications. It was also found that fusing or associating functionaleffector proteins with negatively charged supercharged proteins allowsfor the proteins to associate with cationic lipids or cationic polymers,which provides potent delivery of the proteins to the interior of acell. Further, functional effector proteins that are naturallynegatively charged (e.g., VP64 transcriptional activators, the anionic3×FLAG peptide tag, and fusions thereof) or functional effector proteins(e.g., Cas9 proteins, and variants and fusions thereof) that associatewith nucleic acids (e.g., guide RNAs; “gRNAs”) which are inherentlynegatively charged, can associate with cationic lipids or cationicpolymers for delivery to cells (e.g., in the absence of a superchargedprotein).

While delivery of effector proteins has proven effective forextracellular targets, their use to address intracellular targets iscomparatively undeveloped due to the inability of most proteins tospontaneously enter mammalian cells. Enabling exogenous proteins toaccess intracellular targets is most commonly achieved by delivery oftheir encoding DNA sequences through chemical transfection,electroporation, or viral delivery. The introduction of exogenous DNAinto cells, however, raises the possibility of permanent recombinationinto the genome, potential disruption of endogenous genes, and long-termexposure to the encoded agent. For some research or therapeuticapplications, including genome editing applications that seek to effecta one-time, permanent modification of genomic DNA, the functionaldelivery of non-replicable protein agents may offer improved safety orbroader applicability. Further, while the delivery of proteins usingcationic compounds such as lipids and polymers has remained technicallychallenging and in many cases induces cellular toxicity, it wassurprisingly found, using the compositions and methods provided herein,that certain functional effector proteins (e.g., Cas9 proteins andvariants and fusions thereof, recombinases, transcriptionalactivators/repressors, etc.) can be delivered to cells with no orminimal toxicity, in some cases mediating genomic modifications withsignificant improvements in efficiency and reduced off-target effects.For example, as described in Example 7, delivery of Cas9:gRNA complexeswith cationic lipids is highly efficient (up to 80% modification ofcultured human cells from a single treatment) and also induces highergenome modification specificity compared with plasmid transfection,typically resulting in >10-fold higher on-target:off-target DNAmodification ratios in human cells.

Accordingly, in one aspect, supercharged proteins are used to deliverfunctional effector proteins into cells, for example nucleases,transcriptional activators/repressors, Cas9 proteins (including fusionsand variants thereof), etc. In some embodiments, the superchargedprotein has been engineered to exhibit an increase in its overallsurface charge as compared to the corresponding unmodified protein. Inother embodiments, the supercharged protein has been engineered toexhibit a decrease in its overall surface charge as compared to thecorresponding unmodified protein. In other embodiments, the superchargedprotein used in the context of this disclosure is a naturally occurringsupercharged protein. The supercharged protein may be associated withthe protein to be delivered through covalent or non-covalentinteractions. Without wishing to be bound by any particular theory, theCas9 protein, variant, or fusion protein associated with a gRNA has netnegative charged facilitating association with a positively chargedsupercharged protein. In certain embodiments, the functional effectorprotein associated with the supercharged protein is further associatedwith a cationic polymer or cationic lipid to form a composition suitablefor delivery into a cell. Examples of suitable engineered or naturallyoccurring supercharged proteins are described in international PCTpatent application PCT/US07/70254, filed Jun. 1, 2007, published as WO2007/143574 on Dec. 13, 2007; in international PCT applicationPCT/US09/041,984, filed on Apr. 28, 2009, published as WO 2009/134808 onNov. 5, 2009; and in international PCT application PCT/US10/001,250,filed on Apr. 28, 2010, published as WO 2010/129023 on Nov. 11, 2010;the entire contents of each of which are incorporated herein byreference. Further examples of supercharged proteins for use indelivering nucleases to cells are described herein. Additional examplesof suitable functional effector proteins, for example, nucleases andRNA-programmable effector proteins such as Cas9 proteins, are describedin U.S. Provisional Patent Application Ser. No. 61/868,846, filed Aug.22, 2013, entitled “Engineered Transcription Activator-Like Effector(TALE) Domains and Uses Thereof,” U.S. Provisional Patent ApplicationSer. No. 61/874,609, filed Sep. 6, 2013, entitled “Cas9 Variants andUses Thereof,” U.S. Provisional Patent Application Ser. No. 61/874,682,filed Sep. 6, 2013, entitled “Switchable Cas9 Nucleases and UsesThereof,” U.S. Non-provisional application Ser. No. 14/320,519, filedJun. 20, 2014, entitled “Engineered Transcription Activator-LikeEffector (TALE) Domains and Uses Thereof,” U.S. Non-provisionalapplication Ser. No. 14/320,498, filed Jun. 30, 2014, entitled“Cas9-FokI Fusion Proteins And Uses Thereof,” U.S. Non-provisionalapplication Ser. No. 14/320,467, filed Jun. 30, 2014, entitled“Cas9-Recombinase Fusion Proteins And Uses Thereof,” U.S.Non-provisional application Ser. No. 14/326,329, filed Jul. 8, 2014,entitled “Switchable gRNAs Comprising Aptamers,” U.S. Non-provisionalapplication Ser. No. 14/326,340, filed Jul. 8, 2014, entitled“mRNA-Sensing Switchable gRNAs,” U.S. Non-provisional application Ser.No. 14/326,361, filed Jul. 8, 2014, entitled “Extended DNA-SensinggRNAs,” U.S. Non-provisional application Ser. No. 14/325,815, filed Jul.8, 2014, entitled “Fusions Of Cas9 Domains And Nucleic Acid-EditingDomains,” U.S. Non-provisional application Ser. No. 14/326,109, filedJul. 8, 2014, entitled “Methods For Nucleic Acid Editing,” U.S.Non-provisional application Ser. No. 14/326,140, filed Jul. 8, 2014,entitled “Methods For Correcting PI3K Point Mutations,” U.S.Non-provisional application Ser. No. 14/326,269, filed Jul. 9, 2014,entitled “Methods For Correcting Presenilin Point Mutations,” U.S.Non-provisional application Ser. No. 14/326,290, filed Jul. 8, 2014,entitled “Methods For Correcting α-Antitrypsin Point Mutations,” U.S.Non-provisional application Ser. No. 14/326,318, filed Jul. 8, 2014,entitled “Methods For Correcting Von Willebrand Factor Point Mutations,”U.S. Non-provisional application Ser. No. 14/326,303, filed Jul. 8,2014, entitled “Methods For Correcting Caspase-9 Point Mutations,” andU.S. Provisional Application Ser. No. 62/030,943, entitled “Cas9Proteins Including Ligand-Dependent Inteins,” the entire contents ofeach of which are incorporated herein by reference.

In some embodiments, the supercharged protein, engineered or naturallyoccurring, is positively charged. In other embodiments, for examplethose involving delivery of certain effector proteins using cationiclipids and/or cationic polymers, the supercharged protein is negativelycharged. In certain embodiments, superpositively or supernegativelycharged proteins is non-covalently associated with an effector protein.Alternatively, superpositively or supernegatively charged proteins maybe covalently bound to the effector protein. In some embodiments, theeffector protein is fused to a supercharged protein. In certainembodiments, the resulting fusion protein comprises a linker, e.g., acleavable linker, between the supercharged protein and the effectorprotein.

Some aspects of this disclosure provide compositions comprising asupercharged protein associated with a functional effector protein(e.g., nucleases, transcriptional activators/repressors, recombinases,Cas9 proteins including variants and fusions thereof, etc.). In someembodiments, the composition further comprises a cationic lipid. In someembodiments, the composition further comprises a cationic polymer. Insome embodiments, the composition further comprises a buffer orexcipient. In some embodiments, the supercharged protein has an overallpositive charge that is greater than its corresponding unmodifiedprotein and is in a quantity sufficient for and is formulated fordelivery to and penetration into a cell. In other embodiments, forexample those involving delivery of certain effector proteins usingcationic lipids and/or cationic polymers, the supercharged protein hasan overall negative charge that is greater than its correspondingunmodified protein. In some embodiments, the functional effector proteinis a site-specific enzyme, e.g., a nuclease, Cas9 protein, recombinase,etc. In some embodiments, the Cas9 protein is a wild type Cas9 protein,a Cas9 nickase, or comprises a nuclease inactivated (dCas9) protein. Insome embodiments, the Cas9 protein is a fusion protein comprising dCas9.In some embodiments, the fusion protein comprises a transcriptionalactivator (e.g., VP64), a transcriptional repressor (e.g., KRAB, SID) anuclease domain (e.g., FokI), a recombinase domain (e.g., Hin, Gin, orTn3), a deaminase (e.g., a cytidine deaminase or an adenosine deaminase)or an epigenetic modifier domain (e.g., TET1). In some embodimentsinvolving nucleases, the nuclease is a TALE nuclease, a Cas9 nuclease, aCas9 nickase, or a zinc finger nuclease. In some embodiments, thenuclease specifically binds and cleaves a nucleic acid sequence. In someembodiments, the targeted nucleic acid sequence is a sequence of a genethat is a therapeutic target, for example a gene that is desirable toinactivate in the treatment of a disease. In some embodiments, thetargeted nucleic acid sequence is a PRDM16, PPARγ, VEGF-A, Oct-4, PI3K,presenilin, α-antitrypsin, von willebrand factor, or caspase-9 genesequence.

In some embodiments, the functional effector protein is a transcriptionfactor. In some embodiments, the functional effector protein is a TALEtranscriptional activator or repressor. In some embodiments, thetranscription factor, transcriptional activator, or transcriptionalrepressor specifically binds and activates or represses a gene. In someembodiments, the gene is a therapeutic target. In some embodiments, thefunctional effector protein is a TALE effector. In some embodiments, thesupercharged protein is covalently bound to the functional effectorprotein, thus forming a fusion protein. In some embodiments, thesupercharged protein is associated with the functional effector proteinvia a linker. In some embodiments, the linker is a cleavable linker. Insome embodiments, the linker is a UV-cleavable linker or a linker thatis cleaved by a lysosomal enzyme. In some embodiments, the superchargedprotein is non-covalently associated with the functional effectorprotein, thus forming a complex. In some embodiments, the superchargedprotein has an overall net positive charge. In other embodiments thesupercharged protein has an overall net negative charge, and theprotein(s) are associated with a cationic lipid. In other embodimentsthe supercharged protein has an overall net negative charge, and theprotein(s) are associated with a cationic polymer. In some embodiments,the overall net positive charge is between about +5 and about +40, orthe overall net negative charge is between about −5 and about −50. Insome embodiments, the supercharged protein is more positively charged oris more negatively charged at physiological pH than its correspondingunmodified protein. In some embodiments, the corresponding unmodifiedprotein is a naturally occurring protein. In some embodiments, thesupercharged protein is at least +5 more positively or is at least −5more negatively charged at physiological pH than its correspondingunmodified protein. In some embodiments, the supercharged protein is afluorescent protein. In some embodiments, the supercharged protein isgreen fluorescent protein (GFP). In some embodiments, the superchargedprotein is a superpositively charged GFP. In some embodiments, thesupercharged protein is a superpositively charged GFP (+36 GFP)comprising at least 20 contiguous amino acid residues of the sequence:

(SEQ ID NO: 1) GGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEF VTAAGIKHGRDERYK.In some embodiments, the supercharged protein comprises the amino acidsequence set forth in SEQ ID NO: 1. In some embodiments, thesupercharged protein consists of the amino acid sequence set forth inSEQ ID NO: 1. In some embodiments, the composition is a pharmaceuticalcomposition. In some embodiments, the composition comprises apharmaceutically acceptable excipient. In some embodiments, thecomposition is formulated for administration to a subject and comprisesthe supercharged protein and the functional effector protein in anamount effective for delivery to at least one cell of the subject. Insome embodiments, the composition comprises the supercharged protein andthe functional effector protein in an amount effective for inducing ameasurable therapeutic effect after administration to a subject.

Some aspects of the disclosure provide compositions comprising a Cas9protein associated with a gRNA and a cationic lipid. It was surprisinglyfound that when a Cas9 protein is associated with a gRNA, the complexcan be encapsulated by cationic lipids and effectively delivered tocells. This may be accomplished with or without a supercharged protein.In some embodiments, the composition comprises a Cas9 protein associatedwith a negatively supercharged protein (e.g., supernegatively chargedGFP) and a cationic lipid, which also provides for successful deliveryto a cell. In some embodiments, the composition exhibits low toxicitywhen delivered to a population of cells, for example, wherein at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% of the cells are viablefollowing administration of the composition. In some embodiments, theCas9 protein is a wild type Cas9 protein, a Cas9 nickase, or comprises anuclease inactivated (dCas9) protein. In some embodiments, the Cas9protein is a fusion protein comprising dCas9. In some embodiments, thefusion protein comprises a transcriptional activator (e.g., VP64), atranscriptional repressor (e.g., KRAB, SID) a nuclease domain (e.g.,FokI), a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g.,a cytidine deaminase or an adenosine deaminase) or an epigeneticmodifier domain (e.g., TET1).

Other aspects of the disclosure provide compositions comprising a Cas9protein associated with a gRNA and a cationic polymer. As with cationiclipids, when a Cas9 protein is associated with a gRNA, the complex canassociate with cationic polymers and be effectively delivered to cells.This may be accomplished with or without a supercharged protein. In someembodiments, the composition comprises a Cas9 protein associated with anegatively supercharged protein (e.g., supernegatively charged GFP) anda cationic polymer, which also provides for successful delivery to acell. In some embodiments, the composition exhibits low toxicity whendelivered to a population of cells, for example, wherein at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% of the cells are viablefollowing administration of the composition. In some embodiments, theCas9 protein is a wild type Cas9 protein, a Cas9 nickase, or comprises anuclease inactivated (dCas9) protein. In some embodiments, the Cas9protein is a fusion protein comprising dCas9. In some embodiments, thefusion protein comprises a transcriptional activator (e.g., VP64), atranscriptional repressor (e.g., KRAB, SID) a nuclease domain (e.g.,FokI), a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g.,a cytidine deaminase or an adenosine deaminase) or an epigeneticmodifier domain (e.g., LSD1, TET1).

Some aspects of this disclosure provide methods for administering acomposition provided herein to a subject. In some embodiments, themethod comprises administering a composition described herein to asubject. In some embodiments, the subject is susceptible to, issuffering from, or is displaying one or more symptoms of a disease,disorder, or condition. In some embodiments, the composition isadministered to the subject in an amount sufficient and under suitableconditions for at least one sign or symptom to be ameliorated as aresult of the administration. In some embodiments, the step ofadministering is performed under conditions sufficient for thefunctional effector protein to penetrate a cell of the subject. In someembodiments, the disease, disorder, or condition is associated withabnormally elevated levels of an mRNA, a protein, or combinationthereof. In some embodiments, the composition comprises a nuclease thatspecifically binds and cleaves a genomic sequence, for example, a normalor a pathogenic allele; a gene associated with susceptibility to, oronset or progression of, a disease; a gene encoding a pathogenic RNA orprotein; or a gene encoding an RNA or protein that is expressed atabnormally high levels in diseased cells or tissue. In some embodiments,the step of administering comprises a route of administration selectedfrom the group consisting of oral, intravenous, intramuscular,intra-arterial, subcutaneous, intraventricular, topical, inhalational,and mucosal delivery.

Some aspects of this disclosure provide methods for introducing afunctional effector protein into a cell. In some embodiments, the methodcomprises contacting the cell with a composition comprising asupercharged protein and a functional effector protein as describedherein under conditions suitable for the functional effector protein toenter the cell, thereby introducing the functional effector protein intothe cell. In some embodiments, the method comprises contacting the cellwith a composition comprising a Cas9 protein and a cationic lipid and/orcationic polymer under conditions suitable for the Cas9 protein to enterthe cell, thereby introducing the Cas9 protein into the cell. In someembodiments, the Cas9 protein enters the nucleus of the cell, forexample the Cas9 protein is directed to the nucleus by including anuclear localization signal (NLS) in the protein. In some embodiments,the method further comprises confirming that the functional effectorprotein (e.g., including Cas9) has penetrated the cell. In someembodiments, the cell is in a subject, and the contacting is done invivo. In some embodiments, the subject is diagnosed with having or beingat risk of developing a disease associated with an abnormal expressionlevel of a gene, and wherein the functional effector protein (e.g.,including Cas9) modulates the expression level of the gene. In someembodiments, the method further comprises detecting a change in thelevel of expression of the gene or detecting a therapeutic response inthe subject. In some embodiments, the cell is a somatic cell. In someembodiments, the cell is contacted with the composition or thepharmaceutical composition in an amount, for a time, and underconditions sufficient to induce programming of the cell to a desiredcell fate. In some embodiments, the method further comprises using theprogrammed cell in a cell replacement therapeutic approach. In someembodiments, the cell is a cell carrying a genomic allele associatedwith a disease and the functional effector protein specifically targetsthe allele. In some embodiments, the cell is contacted ex vivo andre-administered to the subject after successful targeting of theundesired allele by the functional effector protein.

Some aspects of this disclosure provide kits comprising a composition asdescribed herein, for example, a composition comprising a superchargedprotein associated with a functional effector protein. In someembodiments, the kits comprises a Cas9 protein and a superchargedprotein. In some embodiments, the kits comprises a Cas9 protein and acationic lipid. In some embodiments, the kits comprises a Cas9 proteinand a cationic polymer. In some embodiments, the kit further comprisesinstructions for using the components included in the kit.

These and other aspects and embodiments of the invention, as well asvarious advantages and utilities will be more apparent with respect tothe drawings and detailed description of the invention.

DEFINITIONS

As used herein and in the claims, the singular forms “a,” “an,” and“the” include the singular and the plural reference unless the contextclearly indicates otherwise. Thus, for example, a reference to “anagent” includes a single agent and a plurality of agents.

The term “associated with” as used herein in the context of two or moremoieties (e.g., proteins or protein domains) refers to the fact that themoieties are physically associated with or connected to one another,either directly or via one or more additional moieties that serve as alinking agent, to form a structure that is sufficiently stable so thatthe moieties remain physically associated under the conditions in whichthe structure is used, e.g., under physiological conditions. Asupercharged protein may be associated with a functional effectorprotein (e.g., nucleases, transcriptional activators/repressors,recombinases, Cas9 proteins including variants and fusions thereof,etc.) through non-covalent interactions (e.g., electrostaticinteractions). In certain embodiments, a supercharged protein may beassociated with a functional effector protein through electrostaticinteractions to form a complex. In some embodiments, a sufficient numberof weaker interactions can provide sufficient stability for moieties toremain physically associated under a variety of different conditions. Incertain embodiments, a supercharged protein is associated with afunctional effector protein via a covalent bond (e.g., an amide bond).In some embodiments, a functional effector protein is associated with asupercharged protein directly by a peptide bond, or indirectly via alinker.

The term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nucleasecomprising a Cas9 protein, or a fragment thereof (e.g., a proteincomprising an active or inactive DNA cleavage domain of Cas9 or apartially inactive DNA cleavage domain (e.g., a Cas9 “nickase”), and/orthe gRNA binding domain of Cas9). In some embodiments, the term “Cas9”refers to a fusion protein comprising Cas9 or a fragment thereof.

In some embodiments, Cas9 refers to Cas9 from: Corynebacterium ulcerans(NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBIRefs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasmataiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref:NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); PsychroflexustorquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacterjejuni (NCBI Ref: YP_002344900.1); or Neisseria. meningitidis (NCBI Ref:YP_002342100.1).

The term “cationic lipid” refers to a lipid which has a cationic, orpositive, charge at physiologic pH. Cationic lipids can take a varietyof forms including, but not limited to, liposomes or micelles. Cationiclipids useful for certain aspects of the present disclosure are known inthe art, and, generally comprise both polar and non-polar domains, bindto polyanions, such as nucleic acid molecules or negatively superchargedproteins, and are typically known to facilitate the delivery of nucleicacids into cells. Examples of useful cationic lipids includepolyethylenimine, polyamidoamine (PAMAM) starburst dendrimers,Lipofectin (a combination of DOTMA and DOPE), Lipofectase,LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000,LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (SynvoluxTherapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead Sciences,Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).Exemplary cationic liposomes can be made fromN-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate(DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol(DC-Chol),2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; anddimethyldioctadecylammonium bromide (DDAB). Cationic lipids have beenused in the art to deliver nucleic acid molecules to cells (see, e.g.,U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099;8,569,256; 8,691,750; 8,748,667; 8,758,810; 8,759,104; 8,771,728; Lewiset al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998.Molecular Membrane Biology 15:1). In addition, other lipid compositionsare also known in the art and include, e.g., those taught in U.S. Pat.No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

The term “cationic polymer,” as used herein, refers to a polymer havinga net positive charge. Cationic polymers are well known in the art, andinclude those described in Samal et al., Cationic polymers and theirtherapeutic potential. Chem Soc Rev. 2012 Nov. 7; 41(21):7147-94; inpublished U.S. patent applications U.S. 2014/0141487 A1, U.S.2014/0141094 A1, U.S. 2014/0044793 A1, U.S. 2014/0018404 A1, U.S.2014/0005269 A1, and U.S. 2013/0344117 A1; and in U.S. Pat. Nos.8,709,466; 8,728,526; 8,759,103; and 8,790,664; the entire contents ofeach are incorporated herein by reference. Exemplary cationic polymersinclude, but are not limited to, polyallylamine (PAH); polyethyleneimine(PEI); poly(L-lysine) (PLL); poly(L-arginine) (PLA); polyvinylaminehomo- or copolymer; a poly(vinylbenzyl-tri-C₁-C₄-alkylammonium salt); apolymer of an aliphatic or araliphatic dihalide and an aliphaticN,N,N′,N′-tetra-C₁-C₄-alkyl-alkylenediamine; a poly(vinylpyridin) orpoly(vinylpyridinium salt); apoly(N,N-diallyl-N,N-di-C₁-C₄-alkyl-ammoniumhalide); a homo- orcopolymer of a quaternized di-C₁-C₄-alkyl-aminoethyl acrylate ormethacrylate; POLYQUAD™; a polyaminoamide; and the like.

The term “deaminase” refers to an enzyme that catalyzes a deaminationreaction. In some embodiments, the deaminase is a cytidine deaminase,catalyzing the hydrolytic deamination of cytidine or deoxycytidine touracil or deoxyuracil, respectively.

The term “effective amount,” as used herein, refers to an amount of abiologically active agent that is sufficient to elicit a desiredbiological response. For example, in some embodiments, an effectiveamount of a functional effector protein (e.g., nucleases,transcriptional activators/repressors, recombinases, Cas9 proteinsincluding variants and fusions thereof, etc.) may refer to the amount ofthe protein that is sufficient to induce a detectable effect (e.g.,cleavage of a target site, modification of a target site, modulation ofgene expression, etc.). Such an effect may be detected in a suitableassay, e.g., in a cell-free assay, or in a target cell, tissue, orsubject organism. As will be appreciated by the skilled artisan, theeffective amount of an agent, e.g., a functional effector protein, mayvary depending on various factors as, for example, on the desiredbiological response, the specific allele to be targeted, the genome,target site, cell, or tissue being targeted, and the superchargedprotein being used.

The term “effector protein” refers to a protein that modulates abiological function of a cell when introduced into the cell, e.g., amodification of a nucleic acid molecule in the cell (such as a cleavage,deamination, recombination, etc.), or a modulation (e.g., increases ordecreases) the expression or the expression level of a gene in the cell.

The term “engineered,” as used herein refers to a protein molecule,complex, substance, or entity that has been designed, produced,prepared, synthesized, and/or manufactured by a human. Accordingly, anengineered product is a product that does not occur in nature. In someembodiments, an engineered protein or composition, e.g., an engineeredsupercharged protein associated with a functional effector protein, suchas a nuclease, Cas9 protein (including variants and fusions thereof) isa supercharged protein that has been designed to meet particularrequirements or to have particular desired features, e.g., to have aspecified net charge, to specifically bind and/or cleave or modify atarget sequence of interest, to have a specific minimal or maximalcleavage or enzymatic activity, and/or to have a specific stability.

The term “epigenetic modifier,” as used herein, refers to a protein orcatalytic domain thereof having enzymatic activity that results in theepigenetic modification of DNA, for example chromosomal DNA. Epigeneticmodifications include, but are not limited to DNA methylation anddemethylation; histone modifications including methylation anddemethylation (e.g., mono-, di- and tri-methylation), histoneacetylation and deacetylation, as well we histone ubiquitylation,phosphorylation, and sumoylation.

The term “functional protein” refers to a protein that is in a form inwhich it exhibits a property and/or activity by which it ischaracterized.

The term “fusion protein” refers to a protein comprising a plurality ofheterologous proteins, protein domains, or peptides, e.g., asupercharged protein and a functional effector protein, associated witheach other via a peptide linkage, thus forming a single amino acidsequence. In certain embodiments, a fusion protein is encoded by a gene.

The term “gene” has its meaning as understood in the art. It will beappreciated by those of ordinary skill in the art that the term “gene”may include gene regulatory sequences (e.g., promoters, enhancers, etc.)and/or intron sequences. It will further be appreciated that definitionsof gene include references to nucleic acids that do not encode proteinsbut rather encode functional RNA molecules such as RNAi agents,ribozymes, tRNAs, etc. For the purpose of clarity it should be notedthat, as used in the present application, the term “gene” generallyrefers to a portion of a nucleic acid that encodes a protein; the termmay optionally encompass regulatory sequences, as will be clear fromcontext to those of ordinary skill in the art. This definition is notintended to exclude application of the term “gene” to non-protein-codingexpression units but rather to clarify that, in most cases, the term asused in this document refers to a protein-coding nucleic acid.

The term “isolated” refers to a molecule, complex, substance, or entitythat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in nature or inan experimental setting), and/or (2) produced, prepared, synthesized,and/or manufactured by a human. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents.

The term “linker,” as used herein, refers to a chemical group or amolecule linking two molecules or moieties, e.g., a supercharged proteinand a nuclease. Typically, the linker is positioned between, or flankedby, two groups, molecules, or other moieties and connected to each onevia a covalent bond, thus connecting the two. In some embodiments, thelinker comprises an amino acid or a plurality of amino acids (e.g., apeptide or protein). In some embodiments, the linker is an organicmolecule, group, polymer, or chemical moiety. In some embodiments, thelinker is a cleavable linker, e.g., the linker comprises a bond that canbe cleaved upon exposure to a cleaving activity, such as UV light or ahydrolytic enzyme, such as a lysosomal protease. In some embodiments,the linker is any stretch of amino acids having at least 1, at least 2,at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,at least 9, at least 10, at least 15, at least 20, at least 25, at least30, at least 40, at least 50, or more amino acids. In some embodiments,the peptide linker comprises repeats of the tri-peptide Gly-Gly-Ser,e.g., comprising the sequence (GGS)_(n), wherein n represents at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats. In some embodiments, thelinker comprises the sequence (GGS)₆ (SEQ ID NO:2). In some embodiments,the peptide linker is the 16 residue “XTEN” linker, or a variant thereof(See, e.g., Schellenberger et al. A recombinant polypeptide extends thein vivo half-life of peptides and proteins in a tunable manner. Nat.Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linkercomprises the sequence SGSETPGTSESATPES (SEQ ID NO:3), SGSETPGTSESA (SEQID NO:4), or SGSETPGTSESATPEGGSGGS (SEQ ID NO:5). In some embodiments,the peptide linker is one or more selected from VPFLLEPDNINGKTC (SEQ IDNO:6), GSAGSAAGSGEF (SEQ ID NO:7), SIVAQLSRPDPA (SEQ ID NO:8),MKIIEQLPSA (SEQ ID NO:9), VRHKLKRVGS (SEQ ID NO:10), GHGTGSTGSGSS (SEQID NO:11), MSRPDPA (SEQ ID NO:12); or GGSM (SEQ ID NO:13).

The term “nuclease,” as used herein, refers to an agent, for example, aprotein or a small molecule, capable of cleaving a phosphodiester bondconnecting nucleotide residues in a nucleic acid molecule. In someembodiments, a nuclease is a protein, e.g., an enzyme that can bind anucleic acid molecule and cleave a phosphodiester bond connectingnucleotide residues within the nucleic acid molecule. A nuclease may bean endonuclease, cleaving a phosphodiester bond within a polynucleotidechain, or an exonuclease, cleaving a phosphodiester bond at the end ofthe polynucleotide chain. In some embodiments, a nuclease is asite-specific nuclease, binding and/or cleaving a specificphosphodiester bond within a specific nucleotide sequence, which is alsoreferred to herein as the “recognition sequence,” the “nuclease targetsite,” or the “target site.” In some embodiments, a nuclease recognizesa single stranded target site, while in other embodiments, a nucleaserecognizes a double-stranded target site, for example, a double-strandedDNA target site. The target sites of many naturally occurring nucleases,for example, many naturally occurring DNA restriction nucleases, arewell known to those of skill in the art. In many cases, a DNA nuclease,such as EcoRI, HindIII, or BamHI, recognize a palindromic,double-stranded DNA target site of 4 to 10 base pairs in length, and cuteach of the two DNA strands at a specific position within the targetsite. Some endonucleases cut a double-stranded nucleic acid target sitesymmetrically, i.e., cutting both strands at the same position so thatthe ends comprise base-paired nucleotides, also referred to herein asblunt ends. Other endonucleases cut a double-stranded nucleic acidtarget site asymmetrically, i.e., cutting each strand at a differentposition so that the ends comprise unpaired nucleotides. Unpairednucleotides at the end of a double-stranded DNA molecule are alsoreferred to as “overhangs,” e.g., as “5′-overhang” or as “3′-overhang,”depending on whether the unpaired nucleotide(s) form(s) the 5′ or the 3′end of the respective DNA strand. Double-stranded DNA molecule endsending with unpaired nucleotide(s) are also referred to as sticky ends,as they can “stick to” other double-stranded DNA molecule endscomprising complementary unpaired nucleotide(s). A nuclease proteintypically comprises a “binding domain” that mediates the interaction ofthe protein with the nucleic acid substrate, and a “cleavage domain”that catalyzes the cleavage of the phosphodiester bond within thenucleic acid backbone. In some embodiments, a nuclease protein can bindand cleave a nucleic acid molecule in a monomeric form, while, in otherembodiments, a nuclease protein has to dimerize or multimerize in orderto cleave a target nucleic acid molecule. Binding domains and cleavagedomains of naturally occurring nucleases, as well as modular bindingdomains and cleavage domains that can be combined to create nucleasesthat bind specific target sites, are well known to those of skill in theart. For example, transcriptional activator like elements can be used asbinding domains to specifically bind a desired target site, and fused orconjugated to a cleavage domain, for example, the cleavage domain ofFokI, to create an engineered nuclease cleaving the desired target site.

The term “nucleic acid” and the term “nucleic acid molecule,” as usedinterchangeably herein, refer to a compound comprising a nucleoside, anucleotide, or a polymer of nucleotides. Typically, polymeric nucleicacids, e.g., nucleic acid molecules comprising three or more nucleotidesare linear molecules, in which adjacent nucleotides are linked to eachother via a phosphodiester linkage. In some embodiments, “nucleic acid”refers to individual nucleic acid residues (e.g. nucleotides and/ornucleosides). In some embodiments, “nucleic acid” refers to anoligonucleotide chain comprising three or more individual nucleotideresidues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably to refer to a polymer ofnucleotides (e.g., a string of at least three nucleotides). In someembodiments, “nucleic acid” encompasses RNA as well as single and/ordouble-stranded DNA. Nucleic acids may be naturally occurring, forexample, in the context of a genome, a transcript, an mRNA, tRNA, rRNA,siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or othernaturally occurring nucleic acid molecule. On the other hand, a nucleicacid molecule may be a non-naturally occurring molecule, e.g., arecombinant DNA or RNA, an artificial chromosome, an engineered genome,or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, orincluding non-naturally occurring nucleotides or nucleosides.Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similarterms include nucleic acid analogs, i.e. analogs having other than aphosphodiester backbone. Nucleic acids can be purified from naturalsources, produced using recombinant expression systems and optionallypurified, chemically synthesized, etc. Where appropriate, e.g., in thecase of chemically synthesized molecules, nucleic acids can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, and backbone modifications. A nucleic acid sequence is presentedin the 5′ to 3′ direction unless otherwise indicated. In someembodiments, a nucleic acid is or comprises natural nucleosides (e.g.adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

The term “pharmaceutical composition,” as used herein, refers to acomposition that can be administrated to a subject, for example, in thecontext of treatment of a disease or disorder. In some embodiments, apharmaceutical composition comprises an active ingredient, e.g., asupercharged protein associated with a functional effector protein, suchas a nuclease, or a nucleic acid encoding a supercharged protein and afunctional effector protein, e.g., in the form of a fusion protein, anda pharmaceutically acceptable excipient.

The term “physiological pH” as used herein refers to a pH value that isfound in a normal, non-pathologic cell or subject. In some embodiments,physiological pH is between pH 5-8. In some embodiments, physiologicalpH is pH 7-7.5, for example, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, orpH 7.5. In some embodiments, physiological pH is pH 6.5-7.5. In someembodiments, physiological pH is pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH7.5, or pH 8.

The term “prevention” or “prevent” refer to the prophylactic treatmentof a subject who is at risk of developing a disease, disorder, orcondition (e.g., at an elevated risk as compared to a control subject,or a control group of subject, or at an elevated risk as compared to theaverage risk of an age-matched and/or gender-matched subject), resultingin a decrease in the probability that the subject will develop thedisease, disorder, or condition (as compared to the probability withoutprevention), and/or to the inhibition of further advancement of analready established disorder.

The term “proliferative disease,” as used herein, refers to any diseasein which cell or tissue homeostasis is disturbed in that a cell or cellpopulation exhibits an abnormally elevated proliferation rate.Proliferative diseases include hyperproliferative diseases, such aspre-neoplastic hyperplastic conditions and neoplastic diseases.Neoplastic diseases are characterized by an abnormal proliferation ofcells and include both benign and malignant neoplasias. Malignantneoplasms are also referred to as cancers.

The term “protein” is interchangeably used herein with the terms“peptide” and “polypeptide” and refers to a polymer of amino acidresidues linked together by peptide (amide) bonds. The terms refer to aprotein, peptide, or polypeptide of any size, structure, or function.Typically, a protein, peptide, or polypeptide will be at least threeamino acids long. A protein, peptide, or polypeptide may refer to anindividual protein or a collection of proteins. One or more of the aminoacids in a protein, peptide, or polypeptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a hydroxyl group, a phosphate group, a farnesyl group, anisofarnesyl group, a fatty acid group, a linker for conjugation,functionalization, or other modification, etc. A protein, peptide, orpolypeptide may also be a single molecule or may be a multi-molecularcomplex. A protein, peptide, or polypeptide may be just a fragment of anaturally occurring protein or peptide. A protein, peptide, orpolypeptide may be naturally occurring, recombinant, or synthetic, orany combination thereof. A protein may comprise different domains, forexample, a TALE effector protein may comprise a nucleic acid bindingdomain and an effector domain, e.g., a nucleic acid cleavage domain or atranscriptional activator or repressor domain. In some embodiments, aprotein comprises a proteinaceous part, e.g., an amino acid sequenceconstituting a nucleic acid binding domain, and an organic compound,e.g., a compound that can act as a nucleic acid cleavage agent.

The term “RNA-programmable nuclease,” and “RNA-guided nuclease” are usedinterchangeably herein and refer to a nuclease that forms a complex with(e.g., binds or associates with) one or more RNA molecule that is not atarget for cleavage. In some embodiments, an RNA-programmable nuclease,when in a complex with an RNA, may be referred to as a nuclease:RNAcomplex. RNA-programmable nucleases include Cas9. Typically, the boundRNA(s) is referred to as a guide RNA (gRNA). gRNAs can exist as acomplex of two or more RNAs, or as a single RNA molecule. gRNAs thatexist as a single RNA molecule may be referred to as single-guide RNAs(sgRNAs), though “gRNA” is used interchangeably to refer to guide RNAsthat exist as either single molecules or as a complex of two or moremolecules. Typically, gRNAs that exist as single RNA species comprisetwo domains: (1) a domain that shares homology to a target nucleic acid(e.g., and directs binding of a Cas9 complex to the target); and (2) adomain that binds a Cas9 protein. The gRNA comprises a nucleotidesequence that complements a target site, which mediates binding of thenuclease/RNA complex to said target site and providing the sequencespecificity of the nuclease:RNA complex.

The term “recombinase,” as used herein, refers to a site-specific enzymethat mediates the recombination of DNA between recombinase recognitionsequences, which results in the excision, integration, inversion, orexchange (e.g., translocation) of DNA fragments between the recombinaserecognition sequences. Recombinases can be classified into two distinctfamilies: serine recombinases (e.g., resolvases and invertases) andtyrosine recombinases (e.g., integrases). Examples of serinerecombinases include, without limitation, Hin, Gin, Tn3, β-six, CinH,ParA, γδ, Bxb1, φC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118,U153, and gp29. Examples of tyrosine recombinases include, withoutlimitation, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine andtyrosine recombinase names stem from the conserved nucleophilic aminoacid residue that the recombinase uses to attack the DNA and whichbecomes covalently linked to the DNA during strand exchange.Recombinases have numerous applications, including the creation of geneknockouts/knock-ins and gene therapy applications. See, e.g., Brown etal, “Serine recombinases as tools for genome engineering.” Methods.2011; 53(4):372-9; Hirano et al., “Site-specific recombinases as toolsfor heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011;92(2):227-39; Chavez and Calos, “Therapeutic applications of the ΦC31integrase system.” Curr. Gene Ther. 2011; 11(5):375-81; Turan and Bode,“Site-specific recombinases: from tag-and-target- totag-and-exchange-based genomic modifications.” FASEB J. 2011;25(12):4088-107; Venken and Bellen, “Genome-wide manipulations ofDrosophila melanogaster with transposons, Flp recombinase, and ΦC31integrase.” Methods Mol. Biol. 2012; 859:203-28; Murphy, “Phagerecombinases and their applications.” Adv. Virus Res. 2012; 83:367-414;Zhang et al., “Conditional gene manipulation: Creating a new biologicalera.” J. Zhejiang Univ. Sci. B. 2012; 13(7):511-24; Karpenshif andBernstein, “From yeast to mammals: recent advances in genetic control ofhomologous recombination.” DNA Repair (Amst). 2012; 1; 11(10):781-8; theentire contents of each are hereby incorporated by reference in theirentirety. The recombinases provided herein are not meant to be exclusiveexamples of recombinases that can be used in embodiments of theinvention. The methods and compositions of the invention can be expandedby mining databases for new orthogonal recombinases or designingsynthetic recombinases with defined DNA specificities (See, e.g., Grothet al., “Phage integrases: biology and applications.” J. Mol. Biol.2004; 335, 667-678; Gordley et al., “Synthesis of programmableintegrases.” Proc. Natl. Acad. Sci. USA. 2009; 106, 5053-5058; theentire contents of each are hereby incorporated by reference in theirentirety). Other examples of recombinases that are useful in the methodsand compositions described herein are known to those of skill in theart, and any new recombinase that is discovered or generated is expectedto be able to be used in the different embodiments of the invention. Insome embodiments, a recombinase (or catalytic domain thereof) is fusedto a Cas9 protein (e.g., dCas9).

The term “recombine,” or “recombination,” in the context of a nucleicacid modification (e.g., a genomic modification), is used to refer tothe process by which two or more nucleic acid molecules, or two or moreregions of a single nucleic acid molecule, are modified by the action ofa recombinase protein. Recombination can result in, inter alia, theinsertion, inversion, excision, or translocation of a nucleic acidsequence, e.g., in or between one or more nucleic acid molecules.

The term “subject,” as used herein, refers to an individual organism. Insome embodiments, the subject is a human of either sex at any stage ofdevelopment. In some embodiments, the subject is a non-human mammal. Insome embodiments, the subject is a non-human primate. In someembodiments, the subject is a rodent. In some embodiments, the subjectis a laboratory animal, for example, a mouse, a rat, a gerbil, a guineapig, a fish, a frog, or a fly. In some embodiments, the subject is afarm animal, for example, a sheep, a goat, a pig, or a cattle. In someembodiments, the subject is a companion animal, for example, a cat or adog. In some embodiments, the subject is a vertebrate, an amphibian, areptile, a fish, an insect, a fly, or a nematode. In some embodiments,the subject is genetically engineered, e.g., a genetically engineerednon-human subject.

The term “supercharge” refers to any modification of a protein thatresults in the increase or decrease of the overall net charge of theprotein. Modifications include, but are not limited to, alterations inamino acid sequence or addition of charged moieties (e.g., carboxylicacid groups, phosphate groups, sulfate groups, amino groups).Supercharging also refers to the association of an agent with a chargedprotein, naturally occurring or modified, to form a complex withincreased or decreased charge relative to the agent alone.

The term “target site,” as used herein in the context of functionaleffector proteins that bind a nucleic acid molecule, such as nucleasesand transcriptional activators or repressors, refers to a sequencewithin a nucleic acid molecule that is bound and acted upon by theeffector protein, e.g., cleaved by the nuclease or transcriptionallyactivated or repressed by the transcriptional activator or repressor,respectively. A target site may be single-stranded or double-stranded.In the context of RNA-guided (e.g., RNA-programmable) nucleases (e.g.,Cas9), a target site typically comprises a nucleotide sequence that iscomplementary to the gRNA of the RNA-programmable nuclease, and aprotospacer adjacent motif (PAM) at the 3′ end adjacent to thegRNA-complementary sequence. For the RNA-guided nuclease Cas9 (orvariants or fusions comprising having gRNA binding activity), the targetsite may be, in some embodiments, 20 base pairs plus a 3 base pair PAM(e.g., NNN, wherein N represents any nucleotide). Typically, the firstnucleotide of a PAM can be any nucleotide, while the two downstreamnucleotides are specified depending on the specific RNA-guided nuclease.Exemplary target sites for RNA-guided nucleases, such as Cas9, are knownto those of skill in the art and include, without limitation, NNG, NGN,NAG, and NGG, wherein N represents any nucleotide. In addition, Cas9nucleases from different species (e.g., S. thermophilus instead of S.pyogenes) recognizes a PAM that comprises the sequence NGGNG. AdditionalPAM sequences are known, including, but not limited to, NNAGAAW and NAAR(see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013),the entire contents of which are incorporated herein by reference). Forexample, the target site of an RNA-guided nuclease, such as, e.g., Cas9,may comprise the structure [NZ]-[PAM], where each N is, independently,any nucleotide, and Z is an integer between 1 and 50, inclusive. In someembodiments, Z is at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, or at least 50. In someembodiments, Z is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In someembodiments, Z is 20. In some embodiments, “target site” may also referto a sequence within a nucleic acid molecule that is bound but notcleaved by a nuclease. For example, certain embodiments described hereinprovide proteins comprising an inactive (or inactivated) Cas9 DNAcleavage domain. Such proteins (e.g., when also including a Cas9 RNAbinding domain) are able to bind the target site specified by the gRNA,however because the DNA cleavage site is inactivated, the target site isnot cleaved by the particular protein. However, such proteins asdescribed herein are typically conjugated, fused, or bound by anotherprotein (e.g., a nuclease, transcriptional activator, recombinase,deaminase, etc.) or molecule that mediates modification of the nucleicacid molecule. In some embodiments, the sequence actually cleaved willdepend on the protein (e.g., nuclease) or molecule that mediatescleavage of the nucleic acid molecule, and in some cases, for example,will relate to the proximity or distance from which the inactivated Cas9protein(s) is/are bound. In the context of nucleases that dimerize, forexample, dimers of a protein comprising an inactive Cas9 (or a Cas9 RNAbinding domain) and a DNA cleavage domain (e.g., FokI cleavage domain oran active Cas9 cleavage domain), a target sites typically comprises aleft-half site (bound by one protein), a right-half site (bound by thesecond protein), and a spacer sequence between the half sites in whichthe cut is made. In some embodiments, either the left-half site or theright half-site (and not the spacer sequence) is cut. This structure([left-half site]-[spacer sequence]-[right-half site]) is referred toherein as an LSR structure. In some embodiments, the left-half siteand/or the right-half site correspond to an RNA-guided target site(e.g., a Cas9 target site). In some embodiments, either or bothhalf-sites are shorter or longer than, e.g., a typical region targetedby Cas9, for example shorter or longer than 20 nucleotides. In someembodiments, the left and right half sites comprise different nucleicacid sequences. In some embodiments, the target site is a sequencecomprising three (3) RNA-guided nuclease target site sequences, forexample, three sequences corresponding to Cas9 target site sequences, inwhich the first and second, and second and third Cas9 target sitesequences are separated by a spacer sequence. In some embodiments, thespacer sequence is at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 125, at least 150, at least 175, at least 200, or atleast 250 bp long.

The terms “transcriptional activator” and “transcriptional repressor”refer to an agent such as a protein (e.g., a transcription factor orfragment thereof), that binds a target nucleic acid sequence and causesan increase or decrease of the level of expression of a gene productassociated with the target nucleic acid sequence, respectively. Forexample, if the target nucleic acid sequence is located within aregulatory region of a gene, a transcriptional activator causes anincrease of the level of expression of a gene product encoded by thegene (conversely, a transcriptional repressor causes a decrease of thelevel of expression of a gene product encoded by the gene). The geneproduct can be an RNA transcribed from the gene (e.g., an mRNA) or apolypeptide translated from an mRNA transcribed from the gene. Typicallyan increase or decrease in the level of an mRNA results in an ordecrease increase in the level of a polypeptide translated therefrom.The level of expression may be determined using standard techniques formeasuring mRNA or protein.

The term “Transcriptional Activator-Like Effector,” (TALE) as usedherein, refers to effector proteins comprising a DNA binding domain,which contains a highly conserved 33-34 amino acid sequence comprising ahighly variable two-amino acid motif (Repeat Variable Diresidue, RVD).The RVD motif determines binding specificity to a nucleic acid sequence,and can be engineered according to methods well known to those of skillin the art to specifically bind a desired DNA sequence (see, e.g.,Miller, Jeffrey; et. al. (February 2011). “A TALE nuclease architecturefor efficient genome editing”. Nature Biotechnology 29 (2): 143-8;Zhang, Feng; et. al. (February 2011). “Efficient construction ofsequence-specific TAL effectors for modulating mammalian transcription”.Nature Biotechnology 29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.;Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J. (2011), Shiu,Shin-Han. ed. “Transcriptional Activators of Human Genes withProgrammable DNA-Specificity”. PLoS ONE 6 (5): e19509; Boch, Jens(February 2011). “TALEs of genome targeting”. Nature Biotechnology 29(2): 135-6; Boch, Jens; et. al. (December 2009). “Breaking the Code ofDNA Binding Specificity of TAL-Type III Effectors”. Science 326 (5959):1509-12; and Moscou, Matthew J.; Adam J. Bogdanove (December 2009). “ASimple Cipher Governs DNA Recognition by TAL Effectors”. Science 326(5959): 1501; the entire contents of each of which are incorporatedherein by reference). The simple relationship between amino acidsequence and DNA recognition has allowed for the engineering of specificDNA binding domains by selecting a combination of repeat segmentscontaining the appropriate RVDs. TALE effector proteins include, withoutlimitation, TALE nucleases (TALENs) and TALE transcriptional activatorsand repressors.

The term “Transcriptional Activator-Like Element Nuclease,” (TALEN) asused herein, refers to an artificial nuclease comprising atranscriptional activator like effector DNA binding domain to a DNAcleavage domain, for example, a FokI domain. A number of modularassembly schemes for generating engineered TALE constructs have beenreported (Zhang, Feng; et. al. (February 2011). “Efficient constructionof sequence-specific TAL effectors for modulating mammaliantranscription”. Nature Biotechnology 29 (2): 149-53; Geiβler, R.;Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J.(2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Geneswith Programmable DNA-Specificity”. PLoS ONE 6 (5): e19509; Cermak, T.;Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller,J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly ofcustom TALEN and other TAL effector-based constructs for DNA targeting”.Nucleic Acids Research; Morbitzer, R.; Elsaesser, J.; Hausner, J.;Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains bymodular cloning”. Nucleic Acids Research; Li, T.; Huang, S.; Zhao, X.;Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B.(2011). “Modularly assembled designer TAL effector nucleases fortargeted gene knockout and gene replacement in eukaryotes”. NucleicAcids Research.; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.;Marillonnet, S. (2011). Bendahmane, Mohammed. ed. “Assembly of DesignerTAL Effectors by Golden Gate Cloning”. PLoS ONE 6 (5): e19722; each ofwhich is incorporated herein by reference).

The term “transcriptional repressor” refers to a transcription factor,e.g., a protein, that binds a target nucleic acid sequence and causes areduction of the level of expression of a gene product associated withthe target nucleic acid sequence. For example, if the target nucleicacid sequence is located within a regulatory region of a gene, atranscriptional repressor causes a reduction of the level of expressionof a gene product encoded by the gene. The gene product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

The term “zinc finger nuclease,” as used herein, refers to a nucleasecomprising a nucleic acid cleavage domain conjugated to a binding domainthat comprises a zinc finger array. In some embodiments, the cleavagedomain is the cleavage domain of the type II restriction endonucleaseFokI. Zinc finger nucleases can be designed to target virtually anydesired sequence in a given nucleic acid molecule for cleavage, and thepossibility to design zinc finger binding domains to bind unique sitesin the context of complex genomes allows for targeted cleavage of asingle genomic site in living cells, for example, to achieve a targetedgenomic alteration of therapeutic value. Targeting a double-strand breakto a desired genomic locus can be used to introduce frame-shiftmutations into the coding sequence of a gene due to the error-pronenature of the non-homologous DNA repair pathway. Zinc finger nucleasescan be generated to target a site of interest by methods well known tothose of skill in the art. For example, zinc finger binding domains witha desired specificity can be designed by combining individual zincfinger motifs of known specificity. The structure of the zinc fingerprotein Zif268 bound to DNA has informed much of the work in this fieldand the concept of obtaining zinc fingers for each of the 64 possiblebase pair triplets and then mixing and matching these modular zincfingers to design proteins with any desired sequence specificity hasbeen described (Pavletich N P, Pabo Colo. (May 1991). “Zinc finger-DNArecognition: crystal structure of a Zif268-DNA complex at 2.1 A”.Science 252 (5007): 809-17, the entire contents of which areincorporated herein). In some embodiments, separate zinc fingers thateach recognizes a 3 base pair DNA sequence are combined to generate 3-,4-, 5-, or 6-finger arrays that recognize target sites ranging from 9base pairs to 18 base pairs in length. In some embodiments, longerarrays are contemplated. In other embodiments, 2-finger modulesrecognizing 6-8 nucleotides are combined to generate 4-, 6-, or 8-zincfinger arrays. In some embodiments, bacterial or phage display isemployed to develop a zinc finger domain that recognizes a desirednucleic acid sequence, for example, a desired nuclease target site of3-30 bp in length. Zinc finger nucleases, in some embodiments, comprisea zinc finger binding domain and a cleavage domain fused or otherwiseconjugated to each other via a linker, for example, a polypeptidelinker. The length of the linker determines the distance of the cut fromthe nucleic acid sequence bound by the zinc finger domain. If a shorterlinker is used, the cleavage domain will cut the nucleic acid closer tothe bound nucleic acid sequence, while a longer linker will result in agreater distance between the cut and the bound nucleic acid sequence. Insome embodiments, the cleavage domain of a zinc finger nuclease has todimerize in order to cut a bound nucleic acid. In some such embodiments,the dimer is a heterodimer of two monomers, each of which comprise adifferent zinc finger binding domain. For example, in some embodiments,the dimer may comprise one monomer comprising zinc finger domain Aconjugated to a FokI cleavage domain, and one monomer comprising zincfinger domain B conjugated to a FokI cleavage domain. In thisnon-limiting example, zinc finger domain A binds a nucleic acid sequenceon one side of the target site, zinc finger domain B binds a nucleicacid sequence on the other side of the target site, and the dimerizeFokI domain cuts the nucleic acid in between the zinc finger domainbinding sites.

The term “zinc finger,” as used herein, refers to a small nucleicacid-binding protein structural motif characterized by a fold and thecoordination of one or more zinc ions that stabilize the fold. Zincfingers encompass a wide variety of differing protein structures (see,e.g., Klug A, Rhodes D (1987). “Zinc fingers: a novel protein fold fornucleic acid recognition”. Cold Spring Harb. Symp. Quant. Biol. 52:473-82, the entire contents of which are incorporated herein byreference). Zinc fingers can be designed to bind a specific sequence ofnucleotides, and zinc finger arrays comprising fusions of a series ofzinc fingers, can be designed to bind virtually any desired targetsequence. Such zinc finger arrays can form a binding domain of aprotein, for example, of a nuclease, e.g., if conjugated to a nucleicacid cleavage domain. Different types of zinc finger motifs are known tothose of skill in the art, including, but not limited to, Cys₂His₂, Gagknuckle, Treble clef, Zinc ribbon, Zn₂/Cys₆, and TAZ2 domain-like motifs(see, e.g., Krishna S S, Majumdar I, Grishin N V (January 2003).“Structural classification of zinc fingers: survey and summary”. NucleicAcids Res. 31 (2): 532-50). Typically, a single zinc finger motif binds3 or 4 nucleotides of a nucleic acid molecule. Accordingly, a zincfinger domain comprising 2 zinc finger motifs may bind 6-8 nucleotides,a zinc finger domain comprising 3 zinc finger motifs may bind 9-12nucleotides, a zinc finger domain comprising 4 zinc finger motifs maybind 12-16 nucleotides, and so forth. Any suitable protein engineeringtechnique can be employed to alter the DNA-binding specificity of zincfingers and/or design novel zinc finger fusions to bind virtually anydesired target sequence from 3-30 nucleotides in length (see, e.g., PaboC O, Peisach E, Grant R A (2001). “Design and selection of novelcys2His2 Zinc finger proteins”. Annual Review of Biochemistry 70:313-340; Jamieson A C, Miller J C, Pabo C O (2003). “Drug discovery withengineered zinc-finger proteins”. Nature Reviews Drug Discovery 2 (5):361-368; and Liu Q, Segal D J, Ghiara J B, Barbas C F (May 1997).“Design of polydactyl zinc-finger proteins for unique addressing withincomplex genomes”. Proc. Natl. Acad. Sci. U.S.A. 94 (11); the entirecontents of each of which are incorporated herein by reference). Fusionsbetween engineered zinc finger arrays and protein domains that cleave anucleic acid can be used to generate a “zinc finger nuclease.” A zincfinger nuclease typically comprises a zinc finger domain that binds aspecific target site within a nucleic acid molecule, and a nucleic acidcleavage domain that cuts the nucleic acid molecule within or inproximity to the target site bound by the binding domain. Typicalengineered zinc finger nucleases comprise a binding domain havingbetween 3 and 6 individual zinc finger motifs and binding target sitesranging from 9 base pairs to 18 base pairs in length. Longer targetsites are particularly attractive in situations where it is desired tobind and cleave a target site that is unique in a given genome.

The terms “treatment,” “treat,” and “treating,” refer to a clinicalintervention aimed to reverse, alleviate, delay the onset of, or inhibitthe progress of a disease or disorder, or one or more symptoms thereof,as described herein. As used herein, the terms “treatment,” “treat,” and“treating” refer to a clinical intervention aimed to reverse, alleviate,delay the onset of, or inhibit the progress of a disease or disorder, orone or more symptoms thereof, as described herein. In some embodiments,treatment may be administered after one or more symptoms have developedand/or after a disease has been diagnosed. In other embodiments,treatment may be administered in the absence of symptoms, e.g., toprevent or delay onset of a symptom or inhibit onset or progression of adisease. For example, treatment may be administered to a susceptibleindividual prior to the onset of symptoms (e.g., in light of a historyof symptoms and/or in light of genetic or other susceptibility factors).Treatment may also be continued after symptoms have resolved, forexample, to prevent or delay their recurrence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic of macromolecular delivery into mammalian cells.

FIG. 2. Programming adipocyte cell fate: the switch from White AdiposeTissue (WAT) to Brown Adipose Tissue (BAT).

FIG. 3. Using supercharged delivery platforms to deliver TALE activatorsprogrammed to target PPARγ or PRDM16.

FIG. 4. Schematic of a fusion protein comprising a +36 GFP fusion, an18.5 mer TALE domain, and a VP64 activation domain.

FIG. 5. Expression and purification of the +36 GFP-TALE activator-fusionprotein.

FIG. 6. Testing for activation of fat cell regulator genes upon deliveryof +36 GFP PPARγ and PRDM16 TALE activator fusion proteins.

FIG. 7. Delivery efficacy of +36 GFP TALE activator fusion proteins atdifferent concentrations.

FIG. 8. Comparison of delivery efficacy of two different +36 GFP-PRDM16TALE fusion proteins in NIH 3T3 cells.

FIG. 9. PPARγ gene expression after delivery of PPARγ-TALE activatorfusion and comparison to various controls.

FIG. 10. PRDM16 gene expression after delivery of RDM16-TALE activatorfusion and comparison to various controls.

FIG. 11. Moderate TALE activity is observed in the presence of serum.

FIG. 12. Validation of viral delivery of PPARγ followed by 7-daytreatment with adipogenesis cocktail.

FIG. 13. Schematic of an assay for programming fibroblasts into WAT andBAT.

FIG. 14. Adipocyte formation observed upon treatment with +36 GFP TALEactivator fusion protein.

FIG. 15. Staining of various treatments after 7 days with LipidTOX redshows formation of adipocytes after viral delivery as well as afterdelivery of supercharged PPARγ TALE activator fusion protein.

FIG. 16. Staining of various treatments after 7 days with LipidTOX redshows formation of adipocytes after viral delivery as well as afterdelivery of supercharged PPARγ TALE activator fusion protein.

FIG. 17. Expression of WAT biomarker genes after viral delivery as wellas after delivery of supercharged PPARγ TALE activator fusion protein.

FIG. 18. Delivery of supercharged PRDM16 TALE activator fusion proteinsto induce brown-fat adipocytes in vivo. Robust adipocyte formation wasobserved after viral delivery of PPARγ and PRDM16 and also afterdelivery of supercharged TALE activator protein fusions.

FIG. 19. Comparison of TALE/TALE, viral/TALE, and viral/viral-inducedexpression of brown fat markers by expression of PPARγ and PRDM16.

FIG. 20. RT-qPCR assessments are consistent with fat celldifferentiation observed by LipidTOX staining.

FIG. 21. Delivery of functional TALE activator fusion proteins ascomplexes with +36 GFP improves TALE activator activity after delivery.

FIG. 22. PRDM16 gene expression after TALE activator fusion deliveryeither as a fusion (+36GFP PRDM16 TALE-3) or a complex (+36GFP+PRDM16TALE-3) with +36GFP. Delivery of complexes tends to increase TALEactivator activity.

FIG. 23. Effect of Aurein peptide fusion to +36GFP on PRDM16 geneexpression after TALE activator fusion delivery (either as a fusion or acomplex with +36GFP).

FIG. 24. PRDM16 gene expression after TALE activator fusion deliveryeither as a fusion (+36GFP PRDM16 TALE-3) or a complex (+36GFP+PRDM16TALE-3) with Lipofectamine LTX.

FIG. 25. Delivery of supercharged fusion proteins or complexes with Cas9into mammalian cells. (GGS)-9-T-ALAL-PKKKRKV corresponds to SEQ IDNO:251.

FIG. 26. Purification of wild-type Cas9 protein and Cas9 fusion proteinswith +36GFP and Aurein-GGS9.

FIG. 27A-B. A strategy for delivering proteins into mammalian cells byfusion or non-covalent complexation with polyanionic macromolecules andencapsulation with cationic lipids is shown. FIG. 27(A) shows thatrecombinases, transcriptional-activator-like effector (TALE) proteins,and Cas9 endonucleases bind nucleic acids and are natively cationic (nettheoretical charges are shown in black) and are not efficientlyencapsulated by cationic lipids. These proteins can be rendered highlyanionic, however, by fusion to either a supernegatively charged proteinsuch as (−30)GFP, or by complexation with polyanionic nucleic acids.FIG. 27(B) shows a schematic representing that cationic lipids commonlyused to transfect DNA and RNA encapsulate the resulting highly anionicproteins or protein:nucleic acid complexes, mediating their deliveryinto mammalian cells.

FIG. 28A-F. Delivery of Cre recombinase to cultured human cells. In FIG.28(A), the fusion of either highly cationic (+36)GFP or highly anionic(−30)GFP to Cre recombinase is shown. A HeLa reporter cell line thatexpresses DsRed upon Cre-mediated recombination was used to evaluate Credelivery efficiency. In FIG. 28(B) HeLa dsRed cells treated with 10 nM(−30)GFP-Cre and the cationic lipid RNAiMAX. Cells were visualized afterincubation for 48 hours in media containing 10% fetal bovine serum(FBS). FIG. 28(C) shows the delivery of (+36)GFP-Cre in 10% FBS media orin serum-free media, and (−30)GFP-Cre with or without the cationic lipidRNAiMAX in full-serum media. FIG. 28(D) presents the effect of cationiclipid dose on functional (−30)GFP-Cre delivery efficacy after 48 hoursin 275 μL media containing 10% FBS. FIG. 28(E) is a comparison ofseveral commercially available cationic lipids and polymers forfunctional delivery efficacy of (−30)dGFP-Cre. FIG. 28(F) shows theRNAiMAX-mediated delivery of multiple anionic peptide or proteinsequences fused to Cre. Error bars reflect the standard deviation fromthree biological replicates performed on different days. (GGS)9corresponds to SEQ ID NO:252 and His6 corresponds to SEQ ID NO:253.

FIG. 29A-B. Delivery of TALE transcriptional activators into culturedhuman cells. FIG. 29(A) shows the design of an 18.5-repeat TALEactivator fused C-terminally to a VP64 activation domain andN-terminally to (−30)GFP and an NLS. The overall net theoretical chargeof the fusion is −43. FIG. 29(B) demonstrates the activation of NTF3transcription by traditional transfection of plasmids encoding TALE-VP64activators that target sites in the NTF3 gene, or by RNAiMAX cationiclipid-mediated delivery of the corresponding NTF3-targeting (−d30)GFP-TALE-VP64 proteins. Gene expression levels were measured byqRT-PCR and are normalized to GAPDH expression levels. Error barsreflect the standard deviation from three biological replicatesperformed on different days. (GGS)₉ corresponds to SEQ ID NO: 252 andHis6 corresponds to SEQ ID NO: 253.

FIG. 30A-E. Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64transcriptional activators to cultured human cells. FIG. 30(A)demonstrates the cationic lipid-mediated delivery of Cas9 proteinvariants complexed with an EGFP-targeting sgRNA or a VEGF-targetingsgRNA to U2OS EGFP reporter cells. Results are compared to that ofstandard transfection of Cas9 and sgRNA expression plasmids. FIG. 30(B)shows the results of a T7 endonuclease I (T7EI) assay to measure themodification of EGFP from no treatment (lane 1), treatment withEGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9protein+VEGF-targeting sgRNA+RNAiMAX (lane 4), transfection of plasmidsexpressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9protein+EGFP-targeting sgRNA+RNAiMAX (lane 6). Indel efficienciescalculated by densitometry are shown below the gel image. FIG. 30(C)presents the results of a T7EI assay of genome modification at EGFP andthree endogenous genes with a single delivery of Cas9 complexed withfour sgRNAs and RNAiMAX. Indel efficiencies calculated by densitometryare shown below the gel image. FIG. 30(D) shows the delivery of Cas9D10A nickase and pairs of sgRNAs either by plasmid transfection or byRNAiMAX-mediated protein:RNA complex delivery. EGFP-disrupting sgRNAsGFP g1+GFP g5, or GFP g3+GFP g7, are expected to result in genedisruption, while GFP g5+GFP g7 target the same strand and are thereforeexpected to be non-functional. FIG. 30(E) shows the delivery ofcatalytically dead (dCas9)-VP64 transcriptional activators that targetNTF3 either by plasmid transfection or RNAiMAX-mediated proteindelivery. Delivery of both VEGF g3 and VEGF g5 sgRNAs served as anegative control for NTF3 gene activation. Error bars reflect thestandard deviation from six biological replicates performed on differentdays.

FIG. 31A-B. The DNA sequence specificity of Cas9-mediated endogenousgene cleavage in cultured human cells by plasmid transfection or bycationic lipid-mediated protein:sgRNA delivery is shown. In FIG. 31(A),a T7EI assay was performed for on-target modification of endogenousCLTA, EMX, and VEGF genes. In FIG. 31(B-D) the on-target:off-target DNAmodification ratio resulting from Cas9:sgRNA for plasmid transfection orcationic lipid-mediated protein:sgRNA delivery is shown. The conditionsfor each treatment were adjusted to result in ˜10% on-target cleavage,enabling a comparison of DNA cleavage specificity between the twodelivery methods under conditions in which on-target gene modificationefficiencies are comparable. P values are listed in Table 2. Each on-and off-target sample was sequenced once with >10,000 sequences analyzedper on-target sample and an average of >111,000 sequences analyzed peroff-target sample (Table 2).

FIGS. 32A-D. The in vivo delivery of Cre recombinase and Cas9:sgRNAcomplexes to hair cells in the mouse inner ear is shown. In FIG. 32(A),the scala media (cochlear duct) of P0 floxP-tdTomato mice (n=4) wereinjected with 0.3 μL of 23 μM (−30)GFP-Cre in 50% RNAiMAX or withRNAiMAX alone (control). After 5 days, tdTomato expression indicative ofCre-mediated recombination was visualized using immunohistology.Red=tdTomato; green=Myo7a; white=Sox2; blue=DAPI. Yellow bracketsindicate the outer hair cell (OHC) layer. FIG. 32(B) shows that, tendays after (−30)GFP-Cre delivery, intact espin (Esp)-expressingstereocilia of tdTomato-positive outer hair cells were present (arrow),similar to stereocilia in control cochlea. Red=tdTomato; green=Esp;white=Sox2; blue=DAPI. FIG. 32(C) is identical to FIG. 32(A) exceptusing Lipofectamine 2000 instead of RNAiMAX. (n=4). The upper and lowerpanels are images of mice cochlea at low and high magnification,respectively, detailing the efficiency of delivery as well as the effecton cochlear architecture and hair cell loss. FIG. 32(D) shows theresults when the scala media (cochlear duct) of P2 Atoh1-GFP mice (n=3)were injected with 0.3 μL of 33 μM Cas9, 33 μM sgRNA in 50% RNAiMAX orLipofectamine 2000. Cas9-mediated gene disruption results in the loss ofGFP expression when visualized 10 days later. The upper panels show GFPsignal only, while lower panels include additional immunohistologicalmarkers. Yellow boxes in the lower panels highlight hair cells that havelost GFP expression. Red=tdTomato; green=Myo7a; white/light blue=Sox2;blue=DAPI. All scale bars, shown in white, are 10 μm.

FIG. 33A-C. Optimization of cationic lipid-mediated delivery of Crerecombinase. FIG. 33(A) shows the optimization of (−30)GFP-Cre deliveryin BSR-TdTomato cells, a second reporter cell line used for measuringCre recombination efficiency. FIG. 33B demonstrates the effect ofRNAiMAX dosage on (−30)GFP-Cre recombination efficiency in HeLa dsRedreporter cells and toxicity as measured by FACS. HeLa cells were sortedby forward-scatter and side-scatter gating to identify live cells thatretained normal morphology. FIG. 33(C) illustrates the relationshipbetween net charge of the protein fused to Cre recombinase and cationiclipid-mediated functional Cre delivery efficiency. Cre recombinase fusedto the domains listed at 25 nM were combined with 1.5 μL RNAiMAX andincubated with HeLa dsRed reporter cells. After 2 days, recombinationefficiency was measured by FACS. Error bars reflect the standarddeviation from three biological replicates performed on different days.

FIGS. 34A-D. Protein uptake by cationic lipid-mediated delivery versussuperpositively charged cationic protein delivery. FIG. 34(A) quantifiesGFP fluorescence from cells treated with either (−30)GFP-Cre and RNAiMAXor (+36)GFP-Cre after washing cells with PBS+heparin (20 U/mL) to removeunbound protein. FIG. 34(B) shows the functional Cre recombinasedelivery efficiency of (−30)GFP-Cre+1.5 μL RNAiMAX relative to Crerecombinase delivery efficiency arising from fusion with (+36)GFP. FIG.34(C) provides a comparison of mCherry uptake by (−30)GFP-fusion+1.5 μMRNAiMAX treatment versus (+36)GFP fusion by measuring mean mCherryfluorescence of total cell population 48 hours after treatment andwashing cells with PBS+heparin. FIG. 34(D) shows the total cellular GFPfluorescence of (−30)GFP-Cre or (+36)GFP-Cre in the presence or absenceof RNAiMAX.

FIG. 35. Delivery optimization of TALE activators designed to target theNTF3 gene. HEK293T cells were treated with either NTF3 TALE plasmid bytransfection of by liposomal delivery of NTF3 TALE proteins. Cells wereharvested after empirically determined optimal incubation time for bothtreatments and analyzed by qRT-PCR for mRNA levels of NTF3. Optimalprotein (25-50 nM) and lipid dosage (1.5 μL RNAiMAX) was chosen forcomparison of two delivery techniques in FIG. 29B. Error bars reflectthe standard deviation from six biological replicates performed ondifferent days.

FIGS. 36A-D. Determination of gene disruption frequency of an EGFPreporter gene by delivery of Cas9:sgRNA and analyzing by flow cytometry.FIG. 36(A) provides a schematic of EGFP disruption in U2OS cells by NHEJinduced by Cas9 double-stranded breaks. FIG. 36(B) shows the delivery ofEGFP-targeting sgRNA or an off-target sgRNA complexed with(−30)dGFP-Cas9 using RNAiMAX along with a plasmid transfection positivecontrol (orange). FIG. 36(C) provides confirmation that disruption ofEGFP fluorescence is not a result of cellular toxicity by treatingsamples with the TO-PRO-3 live/dead stain (Life Technologies, CarlsbadCalif.) and analyzing the resulting cells by flow cytometry. FIG. 36(D)shows testing of the TO-PRO-3 stain by addition of a cellpermeabilizing, but not completely membrane lysing, detergent (0.5%Tween).

FIGS. 37A-D. Optimization of Cas9:sgRNA functional delivery. In FIG.37(A), cationic lipid-mediated delivery efficiency of two testedconstructs shows that the more anionic (−30)dGFP-NLS-Cas9 facilitatesmore efficient delivery at low protein and sgRNA concentrations comparedwith native Cas9. FIG. 37(B) shows the delivery optimization of(−30)dGFP-NLS-Cas9 as a function of protein and sgRNA concentration.FIG. 37(C) shows the delivery of Cas9 protein without any fusions ortags as a function of protein and sgRNA concentration. FIG. 37(D)provides the optimal sgRNA to protein ratio for RNAiMAX-mediateddelivery of (−30)dGFP-NLS-Cas9 and native Cas9. Error bars reflectstandard deviation from three biological replicates performed ondifferent days.

FIGS. 38A-C. The effect of the NLS and/or (−30)dGFP on functional Cas9delivery as a function of both sgRNA and Cas9 concentration. EGFP genedisruption was measured at three fixed sgRNA concentrations: 10 nM (FIG.38(A)), 25 nM (FIG. 38(B)), and 50 nM (FIG. 38(C)), along with varyingprotein concentrations show in the graphs. Delivery was performed using0.8 μL RNAiMAX and assayed by FACS 48 hours later for loss of EGFPfluorescence signal.

FIGS. 39A-C. Effects of RNAiMAX and Lipofectamine 2000 on Cas9:sgRNAdelivery efficiency and cellular toxicity. In FIG. 39(A), EGFP genedisruption at different Cas9 protein concentrations and a constant doseof 100 nM EGFP sgRNA in U2OS EGFP reporter cells treated for 16 hourswith either RNAiMAX or Lipofectamine 2000 is shown. After 16 hours,media was removed and fresh media was added to cells until end point ofassay 48-72 hours post protein delivery treatment. The live cellpopulation was determined by FACS using TO-PRO-3 Live/Dead stain. FIG.39(B) shows the toxicity profile for Cas9:sgRNA delivery to U2OS cellsas a function of Lipofectamine 2000 dose. FIG. 39(C) provides thetoxicity profile for cells as a function of RNAiMAX dose. Error barsreflect standard deviation from three biological replicates performed ondifferent days.

FIG. 40. Optimization of dCas9-VP64 delivery targeting the NTF3 gene atvarying concentrations of protein and sgRNA. HEK293T cells were treatedwith dCas9-VP64 activator and either NTF3-targeting gRNA g2 or a mixtureof all six NTF3-targeting sgRNAs for 16 hours and 0.8 μL RNAiMAX in48-well plate format (275 μL final volume). NTF3 mRNA levels weredetermined by qRT-PCR and normalized to those of GAPDH. Error barsreflect standard deviation from six biological replicates performed ondifferent days.

FIGS. 41A-C. Indel frequencies, measured by high-throughput sequencing,of several human genes treated either by a mock treatment, bytransfection of Cas9 plasmid and sgRNA linear DNA PCR product, or bycationic lipid-mediated protein:sgRNA delivery are depicted. Mocktreatment involved cationic lipid-mediated protein: sgRNA delivery ofEGFP-targeting sgRNA instead of one of the three human gene-targetingsgRNAs. FIG. 41(A) shows the on-target and off-target indel frequenciesfor the CLTA gene. FIG. 41(B) provides the on-target and off-targetindel frequencies for the EMX gene. FIG. 41(C) demonstrates theon-target and off-target indel frequencies for the VEGF gene. Each on-and off-target sample was sequenced once with >10,000 sequences analyzedper on-target sample and an average of >111,000 sequences analyzed peroff-target sample (Table 2).

FIGS. 42A-C. Delivery of Cas9 endonuclease to mouse embryonic stemcells. FIG. 42(A) shows floating spheres treated with Cas9 protein andRNAiMAX but no sgRNA (control) retained strong GFP fluorescence (right),while those treated with Cas9:sgRNA and RNAiMAX exhibited decreased GFPfluorescence (left). Scale bars are 100 μm. FIG. 42(B) shows the controlprogenitor cells after cell attachment, and virtually all the controlprogenitor cells were GFP positive (right panels). Cas9:sgRNA treatmentled to significant reduction in GFP expression (left panels) and manyprogenitor cells showed complete GFP knockdown (arrows) after cellattachment. Scale bars are 20 μm. FIG. 42(C) shows a T7EI assay on stemcells harvested after imaging confirm cleavage of GFP reporter. Similargene target modification efficiencies were observed from cationiclipid-mediated Cas9:sgRNA delivery and transfection of Cas9 and EGFPsgRNA plasmids.

FIGS. 43A-B. Genome modification induced by cationic lipid-mediatedprotein delivery of Cas9 nuclease and sgRNA at endogenous loci in vivo.FIG. 43(A) shows representative examples of genomic DNA sequences at theEGFP on-target locus that are modified following cationic lipid-mediateddelivery of Cas9 and EGFP sgRNA in mouse hair cells. For each exampleshown, the unmodified genomic site is the first sequence, followed bythe most abundant eight sequences containing deletions and threesequences containing insertions. The numbers before each sequenceindicate sequencing counts. The sgRNA target sites are bold andunderlined in green. Insertions and deletions are shown in red. PAM siteis shown in blue. FIG. 43(B) shows an identical analysis as in FIG.42(A) for EMX on-target site in mouse hair cells. The sequences shown inFIG. 43(A), from top to bottom, correspond to SEQ ID NOs:223-236; andthe sequences shown in FIG. 43(B), from top to bottom, correspond to SEQID NOs:237-250.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The present invention provides complexes, compositions, preparations,kits, systems, and related methods for the delivery of functionaleffector proteins, e.g., nucleases, recombinases, and Cas9 proteins(including variants and fusions thereof, e.g., Cas9 nickases and Cas9fusions to deaminases, gene editing enzymes, transcriptional repressorsand activators, epigenetic modifiers, etc.), to a cell by associatingthe functional effector protein with one or more of a superchargedprotein, cationic polymer, and/or cationic lipid. Typically, thefunctional effector protein is delivered to the interior of a cell,e.g., to cause a biological effect in the cell, such as cleavage of agenomic target sequence or modulation of the expression of a targetgene. In some embodiments, the biological effect exerts a therapeuticbenefit to a subject in which the cell is found. The complexes,compositions, preparations, systems, kits, and related methods fordelivery of functional effector proteins are useful for introducing aneffector protein into a cell, e.g., in the context of manipulating thecell for a research or therapeutic purpose. The compositions,preparations, systems, kits, and related methods for delivery offunctional effector proteins provided herein exhibit improved efficacyand reduced cytotoxicity, and ease of preparation as compared to currenttechnologies. The delivery of site-specific proteins, such as TALENs orCas9 proteins (or variants or fusions thereof) using the compositions,preparations, systems, kits, and related methods provided herein allowsfor the targeted manipulation/modification of the genome of a host cellin vitro or in vivo while avoiding the use of more invasive deliverymethods, such as viral delivery of vectors encoding site-specificproteins.

In some embodiments, the inventive technology uses a superchargedprotein to deliver a functional effector protein into a cell. In certainembodiments, the supercharged protein is an engineered protein. In someembodiments, the supercharged protein is a naturally occurringsupercharged protein. Some aspects of this invention are based on therecognition that supercharged proteins are endocytosed by cells; thatfunctional effector proteins that can be associated with superchargedproteins are effectively taken up by cells together with thesupercharged proteins; and that such functional effector proteins retaintheir biological function after cellular uptake, e.g., in that they areable to cleave or modify genomic target sites or modulate transcriptionof a target gene.

In some embodiments, the compositions provided herein comprising asupercharged protein associated with a functional effector protein(e.g., nucleases, transcriptional activators/repressors, recombinases,Cas9 proteins including variants and fusions thereof, etc.) are usefulas therapeutic agents, diagnostic agents, or research tools. In someembodiments, a functional effector protein, such as a nuclease or atranscription factor, may be therapeutically active, e.g., in that ittargets a gene associated with a disease or disorder. In someembodiments, a composition as provided herein, comprising a superchargedprotein and a functional effector protein, such as a nuclease or atranscription factor, is used to modulate the expression of a gene in acell or to modulate a biological pathway (e.g., a signaling pathway, ametabolic pathway) in a cell. In some embodiments, a cell is contactedwith an inventive composition described herein to introduce a functionaleffector protein into the cell. In some embodiments, an inventivecomposition is administered to a subject in need thereof to introduce afunctional effector protein into a cell within the subject, e.g., into acell associated with a disease or disorder. Suitable cells and celltypes for delivery of functional effector proteins according to someaspects of this disclosure include, but are not limited to, human cells,mammalian cells, T-cells, neurons, stem cells, progenitor cells, bloodcells, fibroblasts, epithelial cells, neoplastic cells, and tumor cells.

Supercharged Proteins

Supercharged proteins for use in the present invention can be producedby changing non-conserved amino acids on the surface of a protein tomore polar or charged amino acid residues. In certain embodiments,non-conserved amino acids on the surface of the protein are mutated intoamino acids that are positively charged at physiological pH (pH ˜7.4).The amino acid residues to be modified may be hydrophobic, hydrophilic,charged, or a combination thereof. Supercharged proteins can also beproduced by the attachment of charged moieties to the protein in orderto supercharge the protein. Supercharged proteins frequently areresistant to aggregation, have an increased ability to refold, resistimproper folding, have improved solubility, and are generally morestable under a wide range of conditions, including denaturing conditionssuch as heat or the presence of a detergent.

Supercharged proteins suitable for use according to aspects of thisdisclosure are known in the art and include, without limitation, thosesupercharged proteins disclosed in international PCT patent application,PCT/US07/70254, filed Jun. 1, 2007, published as WO 2007/143574 on Dec.13, 2007; in international PCT application, PCT/US09/041,984, filed onApr. 28, 2009, published as WO 2009/134808 on Nov. 5, 2009; and ininternational PCT application, PCT/US10/001,250, filed on Apr. 28, 2010,published as WO 2010/129023 on Nov. 11, 2010; the entire contents ofeach of which are incorporated herein by reference. In some embodiments,the supercharged protein is an engineered supercharged protein. In someembodiments, the supercharged protein is a naturally occurringsupercharged protein, e.g., a naturally supercharged protein disclosedin international PCT application, PCT/US10/001,250, filed on Apr. 28,2010, published as WO 2010/129023 on Nov. 11, 2010; each of which isincorporated herein by reference. In some embodiments, the superchargedprotein, engineered or naturally occurring, exhibits a charge:molecularweight ratio of greater than 0.8, e.g., ≧0.85, ≧0.9, ≧0.95, ≧1, ≧1.1,≧1.2, ≧1.3, ≧1.4, ≧1.5, ≧1.6, ≧1.7, ≧1.8, ≧1.9, ≧2, ≧2.5, ≧3, ≧4, ≧5,≧6, ≧7, ≧8, or ≧10.

The supercharged protein employed may be derived from any species ofplant, animal, and/or microorganism. In certain embodiments, thesupercharged protein is a mammalian protein. In certain embodiments, thesupercharged protein is a human protein. In certain embodiments, theprotein is derived from an organism typically used in research. Forexample, the protein to be modified may be from a primate (e.g., ape,monkey), rodent (e.g., rabbit, hamster, gerbil), pig, dog, cat, fish(e.g., Danio rerio), nematode (e.g., C. elegans), yeast (e.g.,Saccharomyces cerevisiae), or bacteria (e.g., E. coli). In certainembodiments, the protein is non-immunogenic. In certain embodiments, theprotein is non-antigenic. In certain embodiments, the protein does nothave inherent biological activity or has been modified to have nobiological activity. In certain embodiments, the protein is chosen basedon its targeting ability. In certain embodiments, the protein is a greenfluorescent protein. In some embodiments, the supercharged protein issupercharged glutathione S-transferace (GST). In some embodiments, thesupercharged protein is supercharged streptavidin.

In some embodiments, a supercharged protein is used that has beenmodified to increase the overall net charge, or to increase the totalnumber of charged residues on the protein surface. In certainembodiments, the theoretical net charge of the supercharged protein isincreased by at least +1, at least +2, at least +3, at least +4, atleast +5, at least +10, at least +15, at least +20, at least +25, atleast +30, at least +35, or at least +40 as compared to the unmodifiedprotein. In certain embodiments, the theoretical net charge of thesupercharged protein is at least +1, at least +2, at least +3, at least+4, at least +5, at least +10, at least +15, at least +20, at least +25,at least +30, at least +35, or at least +40 at physiological pH (i.e.,−7.4).

In other embodiments, for example those involving use of cationic lipidsand/or cationic polymers, a supercharged protein is used that has beenmodified to decrease the overall net charge, or to decrease the totalnumber of charged residues on the protein surface. In certainembodiments, the theoretical net charge of the supercharged protein isdecreased (“minus” or “negative” represented by ‘−’) by at least −1, atleast −2, at least −3, at least −4, at least −5, at least −10, at least−15, at least −20, at least −25, at least −30, at least −35, at least−40, at least −45, or at least −50 as compared to the unmodifiedprotein. In certain embodiments, the theoretical net charge of thesupercharged protein is at least −1, at least −2, at least −3, at least−4, at least −5, at least −10, at least −15, at least −20, at least −25,at least −30, at least −35, at least −40, at least −45, or at least −50.

While some exemplary supercharged proteins are described herein in orderto exemplify the inventive technology, the disclosure is not limited inthis respect. Those of skill in the art will be able to ascertainadditional suitable supercharged proteins for delivering functionaleffector proteins to cells based on the instant disclosure. A number ofnaturally occurring proteins may be modified to generate suitablesupercharged proteins. The desired modifications in such proteins may beaccomplished using any techniques known in the art. Recombinant DNAtechniques for introducing such changes in a protein sequence are wellknown in the art. In certain embodiments, the modifications are made bysite-directed mutagenesis of the polynucleotide encoding the protein.Other techniques for introducing mutations are discussed in MolecularCloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, andManiatis (Cold Spring Harbor Laboratory Press: 1989); the treatise,Methods in Enzymology (Academic Press, Inc., N.Y.); Ausubel et al.,Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NewYork, 1999); each of which is incorporated herein by reference.

Supercharged proteins may be further modified. Proteins includingsupercharged proteins can be modified using techniques known to those ofskill in the art. For example, supercharged proteins may be modifiedchemically or biologically. One or more amino acids may be added,deleted, or changed from the primary sequence. For example, apoly-histidine tag or other tag may be added to the supercharged proteinto aid in the purification of the protein. Other peptides or proteinsmay be added onto the supercharged protein to alter the biological,biochemical, and/or biophysical properties of the protein. For example,an endosomolytic peptide may be added to the primary sequence of thesupercharged protein, or a targeting peptide, may be added to theprimary sequence of the supercharged protein. Other modifications of thesupercharged protein include, but are not limited to, post-translationalmodifications (e.g., glycosylation, phosphorylation, acylation,lipidation, farnesylation, acetylation, proteolysis, etc.). In certainembodiments, the supercharged protein is modified to reduce itsimmunogenicity. In certain embodiments, the supercharged protein ismodified to enhance its ability to deliver a functional effector protein(e.g., nucleases, transcriptional activators/repressors, recombinases,Cas9 proteins including variants and fusions thereof, etc.) to a cell.In certain embodiments, the supercharged protein is conjugated to apolymer. For example, the protein may be PEGylated by conjugating theprotein to a polyethylene glycol (PEG) polymer. Other methods can beused to produce supercharged proteins without modification of theprotein sequence. For example, moieties that alter the net charge can beattached to proteins (e.g., by chemical or enzymatic reactions) toprovide surface charge to achieve supercharging. In certain embodiments,the method of modifying proteins described in Shaw et al., ProteinScience 17:1446, 2008 is used to supercharge a protein that is used inthe instantly disclosed inventive technology.

The design and creation of variants of several different superchargedproteins suitable for use with the instantly disclosed technology isdescribed in international PCT patent application, PCT/US07/70254, filedJun. 1, 2007, published as WO 2007/143574 on Dec. 13, 2007; ininternational PCT application, PCT/US09/041,984, filed on Apr. 28, 2009,published as WO 2009/134808 on Nov. 5, 2009; and in international PCTapplication PCT/US10/001,250, filed on Apr. 28, 2010, published as WO2010/129023 on Nov. 11, 2010; the entire contents of each of which areincorporated herein by reference. Some of the disclosed superchargedproteins described therein have been shown to be more stable and toretain their biological function, e.g., their fluorescence in the caseof fluorescent proteins. For example, a green fluorescent protein (GFP)from Aequorea victoria is described in GenBank Accession Number P42212,incorporated herein by reference. The amino acid sequence of this wildtype GFP is as follows:

(SEQ ID NO: 14) MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDELYK

Wild type GFP has a theoretical net charge of −7. Variants with atheoretical net charge of −29, −30, −25, +15, +25, +36, +48, and +49have been reported, e.g., in in international PCT applicationPCT/US10/001,250, filed on Apr. 28, 2010, published as WO 2010/129023 onNov. 11, 2010, the entire contents of which are incorporated herein byreference. Even after heating the +36 GFP to 95° C., 100% of the variantprotein is soluble and the protein retains ≧70% of its fluorescence.

Some aspects of this disclosure are based on the discovery that +36 GFPefficiently delivers functional effector proteins (e.g., nucleases,transcriptional activators/repressors, recombinases, Cas9 proteinsincluding variants and fusions thereof, etc.) to target cells, and thatthe effector proteins so delivered retain their biological function.Therefore, GFP or other proteins with a net charge of at least +15, atleast +25, at least +30, at least +35, or at least +40 are thought to beparticularly useful for introducing functional effector proteins into acell.

In some embodiments, particularly useful supercharged proteins areproteins that allow for a charge distribution or a surface chargedensity similar to that of +36 GFP. Further, in some embodiments,particularly useful supercharged proteins are proteins exhibiting astable folded structure not easily perturbed by supercharging, thusallowing the supercharged protein to be well folded. In someembodiments, particularly useful supercharged proteins are proteinssharing a structural feature with a supercharged protein describedherein or in international PCT patent application, PCT/US07/70254, filedJun. 1, 2007, published as WO 2007/143574 on Dec. 13, 2007; ininternational PCT application, PCT/US09/041,984, filed on Apr. 28, 2009,published as WO 2009/134808 on Nov. 5, 2009; and in international PCTapplication, PCT/US10/001,250, filed on Apr. 28, 2010, published as WO2010/129023 on Nov. 11, 2010; the entire contents of each of which areincorporated herein by reference; for example, a globular structure, ora β-barrel structure. Protein folding, protein fold structure stabilityand perturbation of protein folding by substitution of specific aminoacids with differently charged amino acids, charge distribution, andsurface charge density can be modeled in silico by methods andalgorithms provided herein and others known to those of skill in theart. Accordingly, it will be apparent to those of skill in the art fromno more than routine experimentation, whether a supercharged protein inquestion will be well folded. Thus, those of skill in the art will beable to identify from a given amino acid sequence whether a givensupercharged protein will be useful for cellular delivery of afunctional effector protein according to the technology describedherein.

Some exemplary, suitable variants of GFP include, without limitation:

+15 GFP: (SEQ ID NO: 15) MGHHHHHHGGASKGERLFTGVVPILVELDGDVNGHKFSVRGEGEGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPEGYVQERTISFKKDGTYKTRAEVKFEGRTLVNRIELKGRDFKEKGNILGHKLEYNFNSHNVYITADKRKNGIKANFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSALSKDPKEKRDHMVLLEFVT AAGITHGMDELYK +25 GFP:(SEQ ID NO: 16) MGHHHHHHGGASKGERLFTGVVPILVELDGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGTYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHNVYITADKRKNGIKANFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSALSKDPKEKRDHMVLLEFVT AAGITHGMDELYK +36 GFP:(SEQ ID NO: 17) MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGTVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVT AAGIKHGRDERYK +42 GFP:(SEQ ID NO: 18) MGHHHHHHGGRSKGKRLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVT AAGIKHGRKERYK +48 GFP:(SEQ ID NO: 19) MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVT AAGIKHGRKERYK +49 GFP:(SEQ ID NO: 20) MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLKEFVT AAGIKHGRKERYK (−)30 GFP:(SEQ ID NO: 21) MGHHHHHHGGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVT AAGIDHGMDELYK

It will be apparent to the skilled artisan that the sequences aboveinclude an N-terminal His6 tag, and that sequences without such a tag orwith a different tag are also suitable.

In order to promote the biological function of the functional effectorprotein (e.g., nucleases, transcriptional activators/repressors,recombinases, Cas9 proteins including variants and fusions thereof,etc.) after delivery to a cell, it may be desirable to enhance endosomalescape of the functional effector protein after cellular uptake. Asupercharged protein or a functional effector protein may be fused to orassociated with a protein, peptide, or other entity known to enhanceendosome degradation or lysis of the endosome. In certain embodiments,the peptide is hemagglutinin 2 (HA2) peptide which is known to enhanceendosome degradation. In certain particular embodiments, HA2 peptide isfused to supercharged GFP (e.g., +36 GFP). In certain particularembodiments, the fused protein is of the sequence:

+36 GFP-HA2 (SEQ ID NO: 22)MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKGSAGSAAGSGEFGLFGAIAGFIENGWEGMIDG

In certain embodiments, the endosomolytic peptide is melittin peptide(GIGAVLKVLTTGLPALISWIKRKRQQ, SEQ ID NO: 23) (Meyer et al., JACS 130(11):3272-3273, 2008; which is incorporated herein by reference). In certainembodiments, the melittin peptide is modified by one, two, three, four,or five amino acid substitutions, deletions, and/or additions. Incertain embodiments, the melittin peptide is of the sequence:CIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO:24). In certain particularembodiments, the melittin peptide is fused to supercharged GFP (e.g.,+36 GFP).

In certain embodiments, the endosomolytic peptide is penetratin peptide(RQIKIWFQNRRMKWKK-amide, SEQ ID NO:25), bovine PrP (1-30) peptide(MVKSKIGSWILVLFVAMWSDVGLCKKRPKP-amide, SEQ ID NO: 26), MPGΔ^(NLS)peptide (which lacks a functional nuclear localization sequence becauseof a K→S substitution) (GALFLGWLGAAGSTMGAPKSKRKV, SEQ ID NO:27), TP-10peptide (AGYLLGKINLKALAALAKKIL-amide, SEQ ID NO:28), and/or EB1 peptide(LIRLWSHLIHIWFQNRRLKWKKK-amide, SEQ ID NO:29) (Lundberg et al., 2007,FASEB J. 21:2664; incorporated herein by reference). In certainembodiments, the penetratin, PrP (1-30), MPG, TP-10, and/or EB1 peptideis modified by one, two, three, four, or five amino acid substitutions,deletions, and/or additions. In certain particular embodiments, the PrP(1-30), MPG, TP-10, and/or EB1 peptide is fused to supercharged GFP(e.g., +36 GFP). In some embodiments, an Aurein peptide is fused to thesupercharged protein.

Other peptides or proteins may also be fused to the supercharged proteinor to a fusion protein comprising a supercharged protein and afunctional effector protein (e.g., nucleases, transcriptionalactivators/repressors, recombinases, Cas9 proteins including variantsand fusions thereof, etc.). For example, a targeting peptide may befused to the supercharged protein in order to selectively deliver afunctional effector protein to a particular cell type. Peptides orproteins that enhance cellular uptake of the functional effector proteinmay also be used. In certain embodiments, the peptide fused to thesupercharged protein is a peptide hormone. In certain embodiments, thepeptide fused to the supercharged protein is a peptide ligand.

The exemplary supercharged proteins described in detail herein are notmeant to limit the disclosure, and one of skill in the art willappreciate that other supercharged proteins may be used for the cellulardelivery of functional effector proteins (e.g., nucleases,transcriptional activators/repressors, recombinases, Cas9 proteinsincluding variants and fusions thereof, etc.), including, but notlimited to, other GFP-style fluorescent proteins. In certainembodiments, the supercharged protein is a supercharged version of bluefluorescent protein. In certain embodiments, the supercharged protein isa supercharged version of cyan fluorescent protein. In certainembodiments, the supercharged protein is a supercharged version ofyellow fluorescent protein. Exemplary suitable fluorescent proteinsinclude, but are not limited to, enhanced green fluorescent protein(EGFP), AcGFP, TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire,T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan,mTFP1 (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus,mCitrine, YPet, PhiYFP, ZsYellow1, mBanana, Kusabira Orange, mOrange,dTomato, dTomato-Tandem, DsRed, DsRed2, DsRed-Express (T1),DsRed-Monomer, mTangerine, mStrawberry, AsRed2, mRFP1, JRed, mCherry,HcRedl, mRaspberry, HcRedl, HcRed-Tandem, mPlum, and AQ143.

Yet other proteins that may be supercharged and used, e.g., in thedelivery of functional effector proteins as disclosed herein (e.g.,nucleases, transcriptional activators/repressors, recombinases, Cas9proteins including variants and fusions thereof, etc.), include histonecomponents or histone-like proteins, high-mobility-group proteins(HMGs), enzymes (e.g., amylases, pectinases, hydrolases, proteases,glucose isomerase, lipases, phytases, alglucerase, imiglucerase,agalsidase beta, α-1-iduronidase, acid α-glucosidase, andiduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase.

Charged polymers other than proteins may also be used to deliverfunctional effector proteins. Additionally, as described in greaterdetail herein, cationic lipids and lipid-like materials as well ascationic polymers can also be used to deliver functional effectorproteins. Suitable cationic lipids, lipid-like materials and cationicpolymers are disclosed herein and additional suitable lipids andlipid-like materials are known to those of skill in the art (see, e.g.,those described in Akinc et al., Nature Biotechnology 26, 561-569(2008), the entire contents of which are incorporated herein byreference).

Delivery of Functional Effector Proteins Using Supercharged Proteins

The present invention provides systems and methods for the delivery offunctional effector proteins (e.g., nucleases, transcriptionalactivators/repressors, recombinases, Cas9 proteins including variantsand fusions thereof, etc.) to cells in vivo, ex vivo, or in vitro. Suchsystems and methods typically involve association of the functionaleffector protein with a supercharged protein to form a complex or afusion protein, and delivery of the complex or fusion protein to a cell.In some embodiments, the functional effector protein to be delivered bythe supercharged protein has therapeutic activity. In some embodiments,delivery of the complex or fusion protein to a cell involvesadministering the complex or fusion protein comprising a superchargedprotein associated with a functional effector protein to a subject inneed thereof.

In some embodiments, a functional effector protein (e.g., nucleases,transcriptional activators/repressors, recombinases, Cas9 proteinsincluding variants and fusions thereof, etc.) by itself may not be ableto enter a cell, but is able to enter a cell when associated with asupercharged protein, for example, via a covalent bond or a non-covalentinteraction. In some embodiments, a composition is provided thatincludes a functional effector protein that is covalently bound to asupercharged protein. In some embodiments, the composition includes afunctional effector protein fused to a supercharged protein via apeptide bond, for example, via direct fusion or via a peptide linker. Insome embodiments, the composition includes a functional effector proteinthat is bound to a supercharged protein by non-covalent interaction. Insome embodiments, a supercharged protein is utilized to allow afunctional effector protein to enter a cell. In some embodiments, thefunctional effector protein delivered to the cell associated with asupercharged protein is separated from the supercharged protein afterdelivery to the cell, for example, by cleavage of a linker peptide by acellular protease (e.g., an endosomal protease) or by dissociation ofthe functional effector protein from the supercharged protein in aspecific cellular microenvironment, for example, in the endosome. Insome embodiments, functional effector proteins delivered to a cell by asystem or method provided by this disclosure have therapeutic activity.

In some embodiments, a functional effector protein (e.g., nucleases,transcriptional activators/repressors, recombinases, Cas9 proteinsincluding variants and fusions thereof, etc.) is delivered to a cell invivo, ex vivo, or in vitro by a system, composition, or method providedherein. In some embodiments, a functional effector protein is a proteinable to carry out a biological function within the target cell, forexample, an enzyme able to bind its substrate and to catalyze anenzymatic reaction in the target cell, e.g., a nuclease able to bind andcut a nucleic acid molecule within a target cell, or a transcriptionfactor able to interact with the genome of a target cell and to activateor inhibit transcription of a target gene in the cell.

In some embodiments, a method for generating a fusion of a functionaleffector protein and a supercharged protein includes the generation ofan expression nucleic acid construct containing the coding sequences ofthe functional protein and the supercharged protein, as well as,optionally, a peptide linker, in frame; the expression of such arecombinant fusion protein in a prokaryotic or eukaryotic cell inculture, the extraction and purification of the fusion protein of thefusion protein. In some embodiments, a nucleic acid construct isgenerated in the form of an expression vector, for example, a vectorsuitable for propagation in a bacterial host and for expression in aprokaryotic or eukaryotic cell.

In some embodiments, a vector suitable for fusion protein expression isgenerated by cloning of a nucleotide sequence coding for a functionaleffector protein to be delivered into a cloning vector including anucleotide sequence coding for a supercharged protein under the controlof a eukaryotic and/or a prokaryotic promoter, by a cloning approachthat results in both coding sequences being in frame with each other. Insome embodiments, the cloning vector includes a nucleotide sequencecoding for a peptide linker between a nucleotide sequence coding for asupercharged protein and a restriction site useful for inserting anucleotide sequence coding for a protein in frame with the linker andthe supercharged protein. In some embodiments, the cloning vectorfurther includes an additional sequence enhancing expression of a fusionprotein in a prokaryotic or eukaryotic cell or facilitating purificationof expressed fusion proteins from such cells, for example, a sequencestabilizing a transcript encoding the fusion protein, such as a poly-Asignal, a spliceable intron, a sequence encoding an in-frame peptide orprotein domain tag (e.g., an Arg-tag, calmodulin-binding peptide tag,cellulose-binding domain tag, DsbA tag, c-myc-tag, glutathioneS-transferase tag, FLAG-tag, HAT-tag, His-tag, maltose-binding proteintag, NusA tag, S-tag, SBP-tag, Strep-tag, or thioredoxin tag), or aselection marker or reporter cassette allowing for identification ofcells harboring and expressing the expression construct and/orquantifying the level of expression in such cells. Methods for cloningand expressing fusion proteins are well known to those in the art, see,for example Sambrook et al., Molecular Cloning: A Laboratory Manual,Volume 1-3, CSHL Press (1989); Gellissen et al., Production ofrecombinant proteins, Wiley-VCH, 2005.

In some embodiments, the functional effector protein is associated witha supercharged GFP, for example, +36 GFP or −30 GFP, for delivery to atarget cell. The benefit of endosomal disruption in the delivery ofmacromolecules by supercharged proteins has been previously demonstrated(Wadia et al., Nat. Med. 10, 310-315, 2004) and in some embodiments,additional steps to effect enhanced endosomal escape, as provided hereinor known in the art, are performed. Highly efficient proteininternalization, when coupled with effective endosomal release, has thepotential to minimize the requisite doses of exogenous protein agents,enhancing their potential as research tools and leads for therapeuticdevelopment.

In some embodiments, a composition comprising a functional effectorprotein associated with a supercharged protein is administered to atarget cell after isolation and/or purification. Protein isolationmethods and technologies are well known to those of skill in the art andinclude, for example, affinity chromatography or immunoprecipitation.The methods suitable for isolating and/or purifying a specificfunctional effector proteins, supercharged proteins, and/or fusionproteins will depend on the nature of the respective protein. Forexample, a His-tagged fusion protein can readily be isolated andpurified via Ni or Co ion chromatography, while fusion proteins taggedwith other peptides or domains or untagged fusion proteins can bepurified by other well established methods.

Functional effector proteins suitable for delivery to a target cell invivo, ex vivo, or in vitro, by a system or method provided herein willbe apparent to those of skill in the art and include, for example,DNA-binding proteins, such as transcription factors and nucleases, aswell as Cas9 proteins (including variants and fusions thereof).

In some embodiments, a method, composition, or system provided herein isused to deliver a therapeutic functional effector protein to a cell.Examples of therapeutic proteins include, but are not limited to,nucleases and Cas9 proteins (including variants and fusions thereof)targeting a genomic allele associated with a disease or disorder, andtranscription factors activating a beneficial gene or repressing apathogenic gene.

In some embodiments, Cas9 is fused to a supercharged protein fordelivery to a cell. In some embodiments, the supercharged protein ispositively charged. In some embodiments, the supercharged protein fusedto Cas9 is (+36)GFP. In some embodiments, the fusion of Cas9 and(+36)GFP comprises the amino acid sequence of SEQ ID NO:30 (e.g., withor without a nuclear localization signal (NLS) and with or without a6×His tag), or comprises an amino acid sequence that is at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to the amino acid sequence of SEQ ID NO:30 (e.g., with orwithout a nuclear localization signal (NLS) and with or without a 6×Histag). In some embodiments, the supercharged protein fused to Cas9 is(−30)GFP. In some embodiments, the fusion of Cas9 and (−30)GFP comprisesthe amino acid sequence of SEQ ID NO:31 (e.g., with or without a nuclearlocalization signal (NLS) and with or without a 6×His tag), or comprisesan amino acid sequence that is at least 80%, at least 85%, at least 90%,at least 95%, at least 98%, or at least 99% identical to the amino acidsequence of SEQ ID NO:31 (e.g., with or without a nuclear localizationsignal (NLS) and with or without a 6×His tag).

(+36) dGFP-NLS-Cas9-6xHis (Y67S): (SEQ ID NO: 30)MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLK FICTTGKLPVPWPTLVTTLT SGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GDHHHHHH(−30) dGFP-NLS-Cas9-6xHis (Y67S): (SEQ ID NO: 31)MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLK FICTTGELPVPWPTLVTTLT SGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTALALPKKKRKVMDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GDHHHHHHCompositions of Functional Effector Proteins and Cationic Lipids

Certain aspects of the disclosure relate to the use of cationic lipidsfor the delivery of effector proteins (e.g., nucleases, transcriptionalactivators/repressors, recombinases, Cas9 proteins including variantsand fusions thereof, etc.), for example as opposed to delivering “naked”protein preparations. Surprisingly, existing liposomal delivery reagentsthat have been engineered for the delivery of nucleic acids such as DNAand RNA were found to effectively deliver certain effector proteins(e.g., Cas9 proteins including variants and fusions thereof) both invitro and in vivo, as described herein. Nucleic acid delivery hasbenefited greatly from the development of liposomal reagents over thepast two decades. Cationic liposomal formulations have enabled DNA andRNA transfection to become a routine technique in basic research andhave even been used in clinical trials. The lipid bilayer of theliposome protects encapsulated nucleic acids from degradation and canprevent specific neutralization by antibodies that can bind nakedpreparations of the nucleic acids. Importantly, fusion of the liposomewith the endosomal membrane during endosomal maturation can enablehighly efficient endosomal escape of cationic lipid-delivered cargo.Other non-cationic, but reversibly ionizable, lipid nanoparticleformulations have enabled efficient encapsulation and delivery ofnucleic acids, while avoiding non-specific electrostatic interactionsand consequent sequestration. However, proteins are chemically diverse,and therefore unlike highly anionic nucleic acids, liposomalformulations have not been similarly successful for the efficientdelivery of proteins. For example, while proteins can be encapsulatednon-specifically and delivered by rehydrated lipids in vitro, theefficacy of encapsulation is dependent on protein concentration and isgenerally inefficient, and thus has not seen widespread application.Aspects of the present disclosure relate to the recognition that anionicproteins or protein complexes (including those proteins associated withnucleic acids) may be able to take advantage of the sameelectrostatics-driven encapsulation used by cationic liposomal reagentsfor nucleic acid delivery. While few proteins natively possess thedensity of negative charges found in the phosphate backbone of nucleicacids, translational fusion to, or non-covalent association with, ananionic carrier such as a negatively supercharged protein or a nucleicacid as described herein render the resulting effector protein orprotein complex sufficiently anionic to drive efficient encapsulation ofsuch protein cargoes by cationic liposomal reagents.

In some embodiments, association or fusion with an engineeredsupernegatively charged GFP is capable of driving efficientencapsulation and delivery of proteins into cultured mammalian cells bycationic lipids commonly used to transfect nucleic acids. This approachis effective even at low nanomolar protein concentrations and in thepresence of serum, resulting in up to 1,000-fold more efficientfunctional protein delivery than protein delivery methods that usefusion to cationic peptides or proteins. As shown in the Examples, theefficacy of delivery depends, in some embodiments, on e.g., thetheoretical net charge of the fusion tag, and that popular nativelyanionic peptide tags e.g., 3×FLAG and VP64, can likewise enableliposomal protein delivery.

The Examples further show that Cas9 nuclease protein associated withpolyanionic guide RNAs (gRNA) can be efficiently delivered in functionalform into mammalian cells by these common cationic liposomalformulations because, while not wishing to be bound by any particulartheory, it is believed that the gRNA acts as a polyanionic mediatorbetween the otherwise cationic Cas9 protein and the cationic lipids.Delivery of Cas9:gRNA complexes is not only highly efficient (e.g., upto 80% modification from a single treatment) but also results inmarkedly higher genome modification specificity compared with plasmidtransfection, typically resulting in >10-fold higheron-target:off-target modification ratios, presumably due to thetransient nature of the delivered Cas9:gRNA activity. In someembodiments, delivery of Cas9:gRNA complexes results in at least a2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold or 25-fold orhigher on-target:off-target modification ratio. The Examples alsodemonstrate that this protein delivery approach can be effective invivo, for example by delivering functional Cre recombinase andfunctional Cas9:gRNA complexes to hair cells in the inner ear of mice.

Accordingly, some aspects of the disclosure provide compositionscomprising a Cas9 protein (e.g., as described herein; see e.g., Cas9effector proteins below) and a cationic lipid capable of delivering theCas9 protein to the interior of a cell. In some embodiments, the Cas9protein is associated with a gRNA, which e.g., provides anionic chargeto the complex thereby allowing the Cas9:gRNA complex to be encapsulatedby the cationic lipids. In some embodiments, the Cas9 protein need notbe associated with a gRNA for effective encapsulation by a cationiclipid, but instead is associated with a negatively supercharged protein,as described herein. In some embodiments where a Cas9 protein isassociated with a negatively supercharged protein, the Cas9 protein isalso associated with a gRNA. In some embodiments, the Cas9 protein is awild type Cas9 protein, a fragment of a wild type Cas9 protein, or avariant of a wild type Cas9 protein. In some embodiments, the Cas9protein comprises a dCas9 domain (e.g., as described herein). In someembodiments, the Cas9 protein is a fusion protein comprising a dCas9domain (e.g., as described herein). In some embodiments, the Cas9protein is a Cas9 nickase.

In other embodiments, compositions comprising an effector protein (e.g.,other than a Cas9 protein) and a cationic lipid are provided which arecapable of delivering the effector protein to the interior of a cell(e.g., to the nucleus of the cell). The effector protein is eithernaturally negatively charged, is modified to have a net overall negativecharge, or is associated with a negatively supercharged protein, asdescribed herein. In some embodiments, the effector protein is anyeffector protein described herein. In some embodiments, the effectorprotein is a recombinase, e.g., any recombinase described herein. Insome embodiments, the recombinase is Cre recombinase. In someembodiments, the Cre recombinase comprises the amino acid sequence ofSEQ ID NO:32 (e.g., with or without the 6×His tag). In some embodiments,the Cre recombinase comprises an amino acid sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identical to the amino acid sequence of SEQ ID NO:32 (e.g., with orwithout the 6×His tag). In some embodiments, the Cre recombinase isfused to a supercharged protein (e.g., +36 GFP or −30GFP). In someembodiments, the Cre recombinase fused to a supercharged proteincomprises the amino acid sequence of SEQ ID NO:33 (e.g., with or withoutthe 6×His tag) or SEQ ID NO:34 (e.g., with or without the 6×His tag), orcomprises an amino acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO:33 or SEQ ID NO:34 (e.g., with orwithout the 6×His tag). In some embodiments, the effector protein is aTALE protein, (e.g., as described herein including those provided in theExamples). In some embodiments, the TALE protein comprises one or moreof a VP64 transcriptional activator domain (e.g., SEQ ID NO:35). In someembodiments, the TALE protein with a VP64 transcriptional activatordomain further comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:36-39 (e.g., with or without the 6×His tag). Insome embodiments, the TALE protein with a VP64 transcriptional activatordomain comprises an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO:36-39 (e.g., with or without the 6×His tag). In some embodiments, theTALE effector protein comprises a (−30)GFP domain (e.g., SEQ ID NO:21 orSEQ ID NO:40), a N-terminal region of a TALE domain (e.g., SEQ IDNO:41), a variable repeat domain (e.g., an 18.5mer repeat domain asprovided in Maeder et al., “Robust, synergistic regulation of human geneexpression using TALE activators.” Nat. Methods. 2013; 10, 243-245), aC-terminal TALE domain (e.g., SEQ ID NO:42), a VP64 activation domain(e.g., SEQ ID NO:35), and optionally one or more linkers (e.g., GGS(9),SEQ ID NO:252) between any domain and optionally a sequence tag (e.g.,6×His, SEQ ID NO:253).

While liposomal delivery of cargo such as DNA and RNA has been known toinduce toxicity in targeted cells, it was found that the inventivecompositions described herein deliver their cargo both in vitro and invivo surprisingly with no or low toxicity. For example, in someembodiments, the compositions comprising a Cas9 protein or othereffector proteins described herein exhibit low toxicity whenadministered to a population of cells (e.g., in vitro or in vivo). Insome embodiments, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least99% of the cells in a population are viable following administration ofan inventive composition comprising a Cas9 protein or other effectorprotein and cationic lipids. Methods for assessing the toxicity of acomposition when administered to a population of cells are well known inthe art and include those described in the Examples.

Cre-6xHis (6xHis tag underlined): (SEQ ID NO: 32)MASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH(+36) GFP-Cre-6xHis (+36 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 33)MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH(−30) GFP-Cre-6xHis (−30 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 34)MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGSHHHHHH(+36) GFP-PPARγ-TALE-2 (+36 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 36)MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLHHHHHH(+36) GFP-PRDM16_TALE-3 (+36 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 37)MGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKTGGSGGSGGSGGSGGSGGSGGSGGSGGTAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLHHHHHH(−30) GFP-PPARγ-TALE-2 (−30 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 38)MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLHHHHHH(−30) GFP-PRDM16_TALE-3 (−30 GFP double-underlined; 6xHis tag underlined):(SEQ ID NO: 39)MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYKAPKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPAQVVAIANNNGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPEQVVAIANNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLHHHHHH(−30) GFP: (SEQ ID NO: 40)MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYK N-terminal TALE domain: (SEQ ID NO: 41)APKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL C-terminal TALE domain: (SEQ ID NO: 42)LESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGS VP64 activation domain: (SEQ ID NO: 35)GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLCompositions of Functional Effector Proteins and Cationic Polymers

Certain aspects of the disclosure relate to the use of cationic polymersfor the delivery of effector proteins (e.g., nucleases, transcriptionalactivators/repressors, recombinases, Cas9 proteins including variantsand fusions thereof, etc.), for example as opposed to delivering “naked”protein preparations. As with cationic lipids, aspects of the presentdisclosure relate to the recognition that anionic proteins or proteincomplexes (including those proteins associated with nucleic acids) cantake advantage of electrostatics-driven encapsulation by and/orassociation with cationic polymers for delivery of functional effectorproteins. While few proteins natively possess the density of negativecharges found in the phosphate backbone of nucleic acids, translationalfusion to, or non-covalent association with, an anionic carrier such asa negatively supercharged protein or a nucleic acid as described hereinrender the resulting effector protein or protein complex sufficientlyanionic to drive efficient encapsulation/association of such proteincargoes by cationic polymers.

In some embodiments, association or fusion with an engineeredsupernegatively charged GFP is capable of driving efficientencapsulation/association and delivery of proteins into culturedmammalian cells by cationic polymers. In some embodiments, Cas9 proteinassociated with polyanionic guide RNAs (gRNA) can be efficientlydelivered in functional form into mammalian cells using cationicpolymers. Accordingly, in some embodiments, a composition comprising aCas9 protein and a cationic polymer is provided, wherein the Cas9protein is associated with a gRNA, and the composition is capable ofdelivering the Cas9 protein to the interior of a cell. In someembodiments, delivery of Cas9:gRNA complexes using cationic polymersresults in at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,20-fold or 25-fold or higher on-target:off-target modification ratio ascompared with plasmid transfection of the Cas9 protein.

Accordingly, some aspects of the disclosure provide compositionscomprising a Cas9 protein (e.g., as described herein; see e.g., Cas9effector proteins below) and a cationic polymer capable of deliveringthe Cas9 protein to the interior of a cell. In some embodiments, theCas9 protein is associated with a gRNA, which e.g., provides anioniccharge to the complex thereby allowing the Cas9:gRNA complex to beencapsulated and/or associated with the cationic polymers. In someembodiments, the Cas9 protein need not be associated with a gRNA foreffective encapsulation by and/or association with a cationic lipid, butinstead is associated with a negatively supercharged protein, asdescribed herein. In some embodiments where a Cas9 protein is associatedwith a negatively supercharged protein, the Cas9 protein is alsoassociated with a gRNA. In some embodiments, the Cas9 protein is a wildtype Cas9 protein, a fragment of a wild type Cas9 protein, or a variantof a wild type Cas9 protein. In some embodiments, the Cas9 proteincomprises a dCas9 domain (e.g., as described herein). In someembodiments, the Cas9 protein is a fusion protein comprising a dCas9domain (e.g., as described herein). In some embodiments, the Cas9protein is a Cas9 nickase.

In other embodiments, compositions comprising an effector protein (e.g.,other than a Cas9 protein) and a cationic polymer are provided which arecapable of delivering the effector protein to the interior of a cell(e.g., to the nucleus of the cell). The effector protein is eithernaturally negatively charged, is modified to have a net overall negativecharge, or is associated with a negatively supercharged protein, asdescribed herein. In some embodiments, the effector protein is anyeffector protein described herein. In some embodiments, the effectorprotein is a recombinase, e.g., any recombinase described herein. Insome embodiments, the recombinase is Cre recombinase. In someembodiments, the Cre recombinase comprises the amino acid sequence ofSEQ ID NO:32 (e.g., with or without the 6×His tag). In some embodiments,the Cre recombinase comprises an amino acid sequence that is at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identical to the amino acid sequence of SEQ ID NO:32 (e.g., with orwithout the 6×His tag). In some embodiments, the Cre recombinase isfused to a supercharged protein (e.g., +36 GFP or −30GFP). In someembodiments, the Cre recombinase fused to a supercharged proteincomprises the amino acid sequence of SEQ ID NO:33 (e.g., with or withoutthe 6×His tag) or SEQ ID NO:34 (e.g., with or without the 6×His tag), orcomprises an amino acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99% identical to theamino acid sequence of SEQ ID NO:33 or SEQ ID NO:34 (e.g., with orwithout the 6×His tag). In some embodiments, the effector protein is aTALE protein, (e.g., as described herein including those provided in theExamples). In some embodiments, the TALE protein comprises one or moreof a VP64 transcriptional activator domain (e.g., SEQ ID NO:35). In someembodiments, the TALE protein with a VP64 transcriptional activatordomain further comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:36-39 (e.g., with or without the 6×His tag). Insome embodiments, the TALE protein with a VP64 transcriptional activatordomain comprises an amino acid sequence that is at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO:36-39 (e.g., with or without the 6×His tag). In some embodiments, theTALE effector protein comprises a (−30)GFP domain (e.g., SEQ ID NO:21 orSEQ ID NO:40), a N-terminal region of a TALE domain (e.g., SEQ IDNO:41), a variable repeat domain (e.g., an 18.5mer repeat domain asprovided in Maeder et al., “Robust, synergistic regulation of human geneexpression using TALE activators.” Nat. Methods. 2013; 10, 243-245), aC-terminal TALE domain (e.g., SEQ ID NO:42), a VP64 activation domain(e.g., SEQ ID NO:35), and optionally one or more linkers (e.g., GGS(9),SEQ ID NO:252) between any domain and optionally a sequence tag (e.g.,6×His, SEQ ID NO:253).

In some embodiments, the compositions comprising a Cas9 protein or othereffector proteins described herein and a cationic polymer exhibit lowtoxicity when administered to a population of cells (e.g., in vitro orin vivo). In some embodiments, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, orat least 99% of the cells in a population are viable followingadministration of an inventive composition comprising a Cas9 protein orother effector protein and cationic polymers. Methods for assessing thetoxicity of a composition when administered to a population of cells arewell known in the art and include those described in the Examples.

Cas9 Effector Proteins

In some embodiments, effector proteins comprising a RNA-programmableprotein (or fragment or variant thereof) is delivered to a target cellby a system or method provided herein. In some embodiments, anRNA-guided or RNA-programmable nuclease is delivered to a target cell bya system or method provided herein. In some embodiments, theRNA-programmable protein is a Cas9 nuclease, a Cas9 variant, or a fusionof a Cas9 protein, which is delivered to a target cell by a system ormethod provided herein.

In some embodiments, the RNA-programmable nuclease is a(CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csn1)from Streptococcus pyogenes (see, e.g., “Complete genome sequence of anM1 strain of Streptococcus pyogenes.” Ferretti J. J., McShan W. M.,Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S.,Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G.,Najar F. Z., Ren Q., Zhu H., Song L. expand/collapse author listMcLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y.,Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I.,Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), theentire contents of each of which are incorporated herein by reference.Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNAhybridization to determine target DNA cleavage sites, these proteins areable to cleave, in principle, any sequence specified by the guide RNA.Methods of using RNA-programmable nucleases, such as Cas9, forsite-specific cleavage (e.g., to modify a genome) are known in the art(see e.g., Cong, L. et al. Multiplex genome engineering using CRISPR/Cassystems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided humangenome engineering via Cas9. Science 339, 823-826 (2013); Hwang, W. Y.et al. Efficient genome editing in zebrafish using a CRISPR-Cas system.Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmedgenome editing in human cells. eLife 2, e00471 (2013); Dicarlo, J. E. etal. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cassystems. Nucleic acids research (2013); Jiang, W. et al. RNA-guidedediting of bacterial genomes using CRISPR-Cas systems. Naturebiotechnology 31, 233-239 (2013); the entire contents of each of whichare incorporated herein by reference).

A Cas9 nuclease may also be referred to sometimes as a casn1 nuclease ora CRISPR (clustered regularly interspaced short palindromicrepeat)-associated nuclease. CRISPR is an adaptive immune system thatprovides protection against mobile genetic elements (viruses,transposable elements and conjugative plasmids). CRISPR clusters containspacers, sequences complementary to antecedent mobile elements, andtarget invading nucleic acids. CRISPR clusters are transcribed andprocessed into CRISPR RNA (crRNA). In type II CRISPR systems correctprocessing of pre-crRNA requires a trans-encoded small RNA (tracrRNA),endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA servesas a guide for ribonuclease 3-aided processing of pre-crRNA.Subsequently, Cas9/crRNA/tracrRNA endonucleolytically cleaves linear orcircular dsDNA target complementary to the spacer. The target strandthat is not complementary to the crRNA is first cut endonucleolytically,then trimmed 3′-5′ exonucleolytically. In nature, DNA-binding andcleavage typically requires protein and both RNA. However, single guideRNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporateaspects of both the crRNA and tracrRNA into a single RNA species. Seee.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A.,Charpentier E. Science 337:816-821 (2012), the entire contents of whichis hereby incorporated by reference. Cas9 recognizes a short motif inthe CRISPR repeat sequences (the PAM or protospacer adjacent motif) tohelp distinguish self versus non-self. Cas9 nuclease sequences andstructures are well known to those of skill in the art (see, e.g.,“Complete genome sequence of an M1 strain of Streptococcus pyogenes.”Ferretti J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., LyonK., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S.P., Qian Y., Jia H. G., Najar F. Z., Ren Q., Zhu H., Song L.expand/collapse author list McLaughlin R. E., Proc. Natl. Acad. Sci.U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encodedsmall RNA and host factor RNase III.” Deltcheva E., Chylinski K., SharmaC. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J.,Charpentier E., Nature 471:602-607 (2011); and “A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” JinekM., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E.Science 337:816-821 (2012), the entire contents of each of which areincorporated herein by reference).

Cas9 orthologs have been described in various species, including, butnot limited to, S. pyogenes and S. thermophilus. Additional suitableCas9 nucleases and sequences will be apparent to those of skill in theart based on this disclosure, and such Cas9 nucleases and sequencesinclude Cas9 sequences from the organisms and loci disclosed inChylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families oftype II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737;the entire contents of which are incorporated herein by reference. Insome embodiments, proteins comprising Cas9 proteins or fragments thereofare referred to as “Cas9 variants.” A Cas9 variant shares homology toCas9, or a fragment thereof. For example, a Cas9 variant is at leastabout 70% identical, at least about 80% identical, at least about 90%identical, at least about 95% identical, at least about 98% identical,at least about 99% identical, at least about 99.5% identical, or atleast about 99.9% to wild type Cas9. In some embodiments, the Cas9variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or aDNA-cleavage domain, an N-terminal domain or a C-terminal domain, etc.),such that the fragment is at least about 70% identical, at least about80% identical, at least about 90% identical, at least about 95%identical, at least about 98% identical, at least about 99% identical,at least about 99.5% identical, or at least about 99.9% to thecorresponding fragment of wild type Cas9. In some embodiments, wild typeCas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI ReferenceSequence: NC_017053.1, SEQ ID NO:43 (nucleotide); SEQ ID NO:44 (aminoacid)). In some embodiments, a Cas9 protein has an inactive (e.g., aninactivated) DNA cleavage domain. A nuclease-inactivated Cas9 proteinmay interchangeably be referred to as a “dCas9” protein (for nuclease“dead” Cas9). In some embodiments, dCas9 corresponds to, or comprises inpart or in whole, the amino acid set forth as SEQ ID NO:45, below. Insome embodiments, variants of dCas9 (e.g., variants of SEQ ID NO:45) areprovided. For example, in some embodiments, variants having mutationsother than D10A and H840A are provided, which result in nucleaseinactivated Cas9 (dCas9). Such mutations, by way of example, includeother amino acid substitutions at D10 and H840, or other substitutionswithin the nuclease domain of Cas9 (e.g., substitutions in the HNHnuclease subdomain and/or the RuvC1 subdomain). In some embodiments,variants or homologues of dCas9 (e.g., variants of SEQ ID NO:45) areprovided which are at least about 70% identical, at least about 80%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical, at leastabout 99.5% identical, or at least about 99.9% to SEQ ID NO:45. In someembodiments, variants of dCas9 (e.g., variants of SEQ ID NO:45) areprovided having amino acid sequences which are shorter, or longer thanSEQ ID NO:45, by about 5 amino acids, by about 10 amino acids, by about15 amino acids, by about 20 amino acids, by about 25 amino acids, byabout 30 amino acids, by about 40 amino acids, by about 50 amino acids,by about 75 amino acids, by about 100 amino acids, or more. In someembodiments, Cas9 “nickases” are provided which comprise a mutationwhich inactivates a single nuclease domain in Cas9. Such nickases inducea single strand break in a target nucleic acid as opposed to a doublestrand break.

Cas9 (SEQ ID NO: 43)ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGATTATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGGCAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGCAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAATCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTAGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAGAAATGGCTTGTTTGGGAATCTCATTGCTTTGTCATTGGGATTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATAGTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAGCGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAGGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGCGCCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGGGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGATATTCAAAAAGCACAGGTGTCTGGACAAGGCCATAGTTTACATGAACAGATTGCTAACTTAGCTGGCAGTCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAATTGTTGATGAACTGGTCAAAGTAATGGGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTACAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCATTAAAGACGATTCAATAGACAATAAGGTACTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA (SEQ ID NO: 44)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(single underline: HNH domain; double underline: RuvC domain)dCas9 (D10A and H840A): (SEQ ID NO: 45)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(single underline: HNH domain; double underline: RuvC domain)Cas9 nickase (D10A): (SEQ ID NO: 46)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

In some embodiments, fusion proteins comprising a Cas9 protein areprovided for use in any of the compositions and methods describedherein. In some embodiments, the fusion protein comprises a dCas9protein (e.g., as described herein). In some embodiments, the fusionprotein comprises a linker (e.g., as described herein) between dCas9 andone or more domains (e.g., enzymatic domains). In some embodiments, thefusion protein comprises dCas9 and a transcriptional activator domain, atranscriptional repressor domain, a recombinase domain, a gene editingdomain (e.g., a deaminase doman), or an epigenetic modifier domain.

In some embodiments, the general architecture of exemplary fusionproteins provided herein comprises the structure:[NH₂]-[enzymatic domain]-[dCas9]-[COOH] or[NH₂]-[dCas9]-[enzymatic domain]-[COOH];wherein NH₂ is the N-terminus of the fusion protein, COOH is theC-terminus of the fusion protein, and the enzymatic domain comprises anuclease domain (e.g., FokI), a recombinase catalytic domain (e.g., Hin,Gin, or Tn3 recombinase domains), a nucleic acid-editing domain (e.g., adeaminase domain), a transcriptional activator domain (e.g., VP64, p65),a transcriptional repressor domain (e.g., KRAB, SID), or an epigeneticmodifier (e.g., LSD1 histone demethylase, TET1 hydroxylase).

Additional features may be present, for example, one or more linkersequences between certain domains. Other exemplary features that may bepresent are localization sequences, such as nuclear localizationsequences (NLS; e.g., MAPKKKRKVGIHRGVP (SEQ ID NO:47)); cytoplasmiclocalization sequences; export sequences, such as nuclear exportsequences; or other localization sequences, as well as sequence tagsthat are useful for solubilization, purification, or detection of thefusion proteins. Suitable localization signal sequences and sequences ofprotein tags are provided herein and are known in the art, and include,but are not limited to, biotin carboxylase carrier protein (BCCP) tags,myc-tags, calmodulin-tags, FLAG-tags (e.g., 3×FLAG TAG:MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO:48)), hemagglutinin (HA) tags,polyhistidine tags, also referred to as histidine tags or His-tags,maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase(GST) tags, green fluorescent protein (GFP) tags, thioredoxin-tags,S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligasetags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable sequenceswill be apparent to those of skill in the art.

In some embodiments, the enzymatic domain comprises a nuclease or acatalytic domain thereof. For example, in some embodiments, the generalarchitecture of exemplary ligand-dependent dCas9 fusion proteins with anuclease domain comprises the structure:[NH₂]-[NLS]-[dCas9]-[nuclease]-[COOH],[NH₂]-[NLS]-[nuclease]-[dCas9]-[COOH],[NH₂]-[dCas9]-[nuclease]-[COOH], or[NH₂]-[nuclease]-[dCas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, a linker is inserted between the dCas9 and thenuclease domain. In some embodiments, a linker is inserted between theNLS and the nuclease and/or dCas9 domain. In some embodiments, the NLSis located C-terminal of the nuclease and/or the dCas9 domain. In someembodiments, the NLS is located between the nuclease and the dCas9domain. Additional features, such as sequence tags, may also be present.In some aspects, the nuclease domain is a nuclease requiringdimerization (e.g., the coming together of two monomers of the nuclease)in order to cleave a target nucleic acid (e.g., DNA). In someembodiments, the nuclease domain is a monomer of the FokI DNA cleavagedomain. The FokI DNA cleavage domain is known, and in some aspectscorresponds to amino acids 388-583 of FokI (NCBI accession numberJ04623). In some embodiments, the FokI DNA cleavage domain correspondsto amino acids 300-583, 320-583, 340-583, or 360-583 of FokI. See alsoWah et al., “Structure of FokI has implications for DNA cleavage” Proc.Natl. Acad. Sci. USA. 1998; 1; 95(18):10564-9; Li et al., “TAL nucleases(TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavagedomain”Nucleic Acids Res. 2011; 39(1):359-72; Kim et al., “Hybridrestriction enzymes: zinc finger fusions to Fok I cleavage domain” Proc.Natl. Acad. Sci. USA. 1996; 93:1156-1160; the entire contents of eachare herein incorporated by reference). In some embodiments, the FokI DNAcleavage domain corresponds to, or comprises in part or whole, the aminoacid sequence set forth as SEQ ID NO:49. In some embodiments, the FokIDNA cleavage domain is a variant of FokI (e.g., a variant of SEQ IDNO:49), as described herein. Other exemplary compositions and methods ofusing dCas9-nuclease fusion proteins can be found in U.S. patentapplication Ser. No. 14/320,498; titled “Cas9-FokI fusion Proteins andUses Thereof,” filed Jun. 30, 2014; the entire contents of which areincorporated herein by reference.

FokI nuclease domain: (SEQ ID NO: 49)GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFfCas9 (e.g., dCas9-NLS-GGS3linker-FokI): (SEQ ID NO: 50)ATGGATAAAAAGTATTCTATTGGTTTAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGATCAGGTGGAAGTGGCGGCAGCGGAGGTTCTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTfCas9 (e.g., NLS-dCas9-GGS3linker-FokI): (SEQ ID NO: 51)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTATGGATAAAAAGTATTCTATTGGTTTAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACTCAGGTGGAAGTGGCGGCAGCGGAGGTTCTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTfCas9 (e.g., FokI-GGS3linker-dCas9-NLS): (SEQ ID NO: 52)ATGGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTGGCGGTAGTGGGGGATCTGGGGGAAGTATGGATAAAAAGTATTCTATTGGTTTAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGACGGATCCCCCAAGAAGAAGAGGAAAGTCTCGAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCTGCAGGAfCas9 (e.g., NLS-FokI-GGS3linker-dCas9): (SEQ ID NO: 53)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTGGAGGTTCTATGGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTGGCGGTAGTGGGGGATCTGGGGGAAGTATGGATAAAAAGTATTCTATTGGTTTAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGAC fCas9: (SEQ ID NO: 54)ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGATGGCCCCCAAGAAGAAGAGGAAGGTGGGCATTCACCGCGGGGTACCTGGAGGTTCTGGATCCCAACTAGTCAAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATTCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCGAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTGTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCCGGCACATTAACCTTAGAGGAAGTCAGACGGAAATTTAATAACGGCGAGATAAACTTTAGCGGCAGCGAGACTCCCGGGACCTCAGAGTCCGCCACACCCGAAAGTGATAAAAAGTATTCTATTGGTTTAGCTATCGGCACTAATTCCGTTGGATGGGCTGTCATAACCGATGAATACAAAGTACCTTCAAAGAAATTTAAGGTGTTGGGGAACACAGACCGTCATTCGATTAAAAAGAATCTTATCGGTGCCCTCCTATTCGATAGTGGCGAAACGGCAGAGGCGACTCGCCTGAAACGAACCGCTCGGAGAAGGTATACACGTCGCAAGAACCGAATATGTTACTTACAAGAAATTTTTAGCAATGAGATGGCCAAAGTTGACGATTCTTTCTTTCACCGTTTGGAAGAGTCCTTCCTTGTCGAAGAGGACAAGAAACATGAACGGCACCCCATCTTTGGAAACATAGTAGATGAGGTGGCATATCATGAAAAGTACCCAACGATTTATCACCTCAGAAAAAAGCTAGTTGACTCAACTGATAAAGCGGACCTGAGGTTAATCTACTTGGCTCTTGCCCATATGATAAAGTTCCGTGGGCACTTTCTCATTGAGGGTGATCTAAATCCGGACAACTCGGATGTCGACAAACTGTTCATCCAGTTAGTACAAACCTATAATCAGTTGTTTGAAGAGAACCCTATAAATGCAAGTGGCGTGGATGCGAAGGCTATTCTTAGCGCCCGCCTCTCTAAATCCCGACGGCTAGAAAACCTGATCGCACAATTACCCGGAGAGAAGAAAAATGGGTTGTTCGGTAACCTTATAGCGCTCTCACTAGGCCTGACACCAAATTTTAAGTCGAACTTCGACTTAGCTGAAGATGCCAAATTGCAGCTTAGTAAGGACACGTACGATGACGATCTCGACAATCTACTGGCACAAATTGGAGATCAGTATGCGGACTTATTTTTGGCTGCCAAAAACCTTAGCGATGCAATCCTCCTATCTGACATACTGAGAGTTAATACTGAGATTACCAAGGCGCCGTTATCCGCTTCAATGATCAAAAGGTACGATGAACATCACCAAGACTTGACACTTCTCAAGGCCCTAGTCCGTCAGCAACTGCCTGAGAAATATAAGGAAATATTCTTTGATCAGTCGAAAAACGGGTACGCAGGTTATATTGACGGCGGAGCGAGTCAAGAGGAATTCTACAAGTTTATCAAACCCATATTAGAGAAGATGGATGGGACGGAAGAGTTGCTTGTAAAACTCAATCGCGAAGATCTACTGCGAAAGCAGCGGACTTTCGACAACGGTAGCATTCCACATCAAATCCACTTAGGCGAATTGCATGCTATACTTAGAAGGCAGGAGGATTTTTATCCGTTCCTCAAAGACAATCGTGAAAAGATTGAGAAAATCCTAACCTTTCGCATACCTTACTATGTGGGACCCCTGGCCCGAGGGAACTCTCGGTTCGCATGGATGACAAGAAAGTCCGAAGAAACGATTACTCCATGGAATTTTGAGGAAGTTGTCGATAAAGGTGCGTCAGCTCAATCGTTCATCGAGAGGATGACCAACTTTGACAAGAATTTACCGAACGAAAAAGTATTGCCTAAGCACAGTTTACTTTACGAGTATTTCACAGTGTACAATGAACTCACGAAAGTTAAGTATGTCACTGAGGGCATGCGTAAACCCGCCTTTCTAAGCGGAGAACAGAAGAAAGCAATAGTAGATCTGTTATTCAAGACCAACCGCAAAGTGACAGTTAAGCAATTGAAAGAGGACTACTTTAAGAAAATTGAATGCTTCGATTCTGTCGAGATCTCCGGGGTAGAAGATCGATTTAATGCGTCACTTGGTACGTATCATGACCTCCTAAAGATAATTAAAGATAAGGACTTCCTGGATAACGAAGAGAATGAAGATATCTTAGAAGATATAGTGTTGACTCTTACCCTCTTTGAAGATCGGGAAATGATTGAGGAAAGACTAAAAACATACGCTCACCTGTTCGACGATAAGGTTATGAAACAGTTAAAGAGGCGTCGCTATACGGGCTGGGGACGATTGTCGCGGAAACTTATCAACGGGATAAGAGACAAGCAAAGTGGTAAAACTATTCTCGATTTTCTAAAGAGCGACGGCTTCGCCAATAGGAACTTTATGCAGCTGATCCATGATGACTCTTTAACCTTCAAAGAGGATATACAAAAGGCACAGGTTTCCGGACAAGGGGACTCATTGCACGAACATATTGCGAATCTTGCTGGTTCGCCAGCCATCAAAAAGGGCATACTCCAGACAGTCAAAGTAGTGGATGAGCTAGTTAAGGTCATGGGACGTCACAAACCGGAAAACATTGTAATCGAGATGGCACGCGAAAATCAAACGACTCAGAAGGGGCAAAAAAACAGTCGAGAGCGGATGAAGAGAATAGAAGAGGGTATTAAAGAACTGGGCAGCCAGATCTTAAAGGAGCATCCTGTGGAAAATACCCAATTGCAGAACGAGAAACTTTACCTCTATTACCTACAAAATGGAAGGGACATGTATGTTGATCAGGAACTGGACATAAACCGTTTATCTGATTACGACGTCGATGCCATTGTACCCCAATCCTTTTTGAAGGACGATTCAATCGACAATAAAGTGCTTACACGCTCGGATAAGAACCGAGGGAAAAGTGACAATGTTCCAAGCGAGGAAGTCGTAAAGAAAATGAAGAACTATTGGCGGCAGCTCCTAAATGCGAAACTGATAACGCAAAGAAAGTTCGATAACTTAACTAAAGCTGAGAGGGGTGGCTTGTCTGAACTTGACAAGGCCGGATTTATTAAACGTCAGCTCGTGGAAACCCGCCAAATCACAAAGCATGTTGCACAGATACTAGATTCCCGAATGAATACGAAATACGACGAGAACGATAAGCTGATTCGGGAAGTCAAAGTAATCACTTTAAAGTCAAAATTGGTGTCGGACTTCAGAAAGGATTTTCAATTCTATAAAGTTAGGGAGATAAATAACTACCACCATGCGCACGACGCTTATCTTAATGCCGTCGTAGGGACCGCACTCATTAAGAAATACCCGAAGCTAGAAAGTGAGTTTGTGTATGGTGATTACAAAGTTTATGACGTCCGTAAGATGATCGCGAAAAGCGAACAGGAGATAGGCAAGGCTACAGCCAAATACTTCTTTTATTCTAACATTATGAATTTCTTTAAGACGGAAATCACTCTGGCAAACGGAGAGATACGCAAACGACCTTTAATTGAAACCAATGGGGAGACAGGTGAAATCGTATGGGATAAGGGCCGGGACTTCGCGACGGTGAGAAAAGTTTTGTCCATGCCCCAAGTCAACATAGTAAAGAAAACTGAGGTGCAGACCGGAGGGTTTTCAAAGGAATCGATTCTTCCAAAAAGGAATAGTGATAAGCTCATCGCTCGTAAAAAGGACTGGGACCCGAAAAAGTACGGTGGCTTCGATAGCCCTACAGTTGCCTATTCTGTCCTAGTAGTGGCAAAAGTTGAGAAGGGAAAATCCAAGAAACTGAAGTCAGTCAAAGAATTATTGGGGATAACGATTATGGAGCGCTCGTCTTTTGAAAAGAACCCCATCGACTTCCTTGAGGCGAAAGGTTACAAGGAAGTAAAAAAGGATCTCATAATTAAACTACCAAAGTATAGTCTGTTTGAGTTAGAAAATGGCCGAAAACGGATGTTGGCTAGCGCCGGAGAGCTTCAAAAGGGGAACGAACTCGCACTACCGTCTAAATACGTGAATTTCCTGTATTTAGCGTCCCATTACGAGAAGTTGAAAGGTTCACCTGAAGATAACGAACAGAAGCAACTTTTTGTTGAGCAGCACAAACATTATCTCGACGAAATCATAGAGCAAATTTCGGAATTCAGTAAGAGAGTCATCCTAGCTGATGCCAATCTGGACAAAGTATTAAGCGCATACAACAAGCACAGGGATAAACCCATACGTGAGCAGGCGGAAAATATTATCCATTTGTTTACTCTTACCAACCTCGGCGCTCCAGCCGCATTCAAGTATTTTGACACAACGATAGATCGCAAACGATACACTTCTACCAAGGAGGTGCTAGACGCGACACTGATTCACCAATCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGAC

In some embodiments, the enzymatic domain comprises a recombinase orcatalytic domain thereof. For example, in some embodiments, the generalarchitecture of exemplary ligand-dependent dCas9 fusion proteins with arecombinase domain comprises the structure:[NH₂]-[NLS]-[dCas9]-[recombinase]-[COOH],[NH₂]-[NLS]-[recombinaseHdCas9HCOOH],[NH₂]-[dCas9]-[recombinase]-[COOH], or[NH₂]-[recombinase]-[dCas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, a linker is inserted between the dCas9 and therecombinase domain. In some embodiments, a linker is inserted betweenthe NLS and the recombinase and/or dCas9 domain. In some embodiments,the NLS is located C-terminal of the recombinase domain and/or the dCas9domain. In some embodiments, the NLS is located between the recombinasedomain and the dCas9 domain. Additional features, such as sequence tags,may also be present. By “catalytic domain of a recombinase,” it is meantthat a fusion protein includes a domain comprising an amino acidsequence of (e.g., derived from) a recombinase, such that the domain issufficient to induce recombination when contacted with a target nucleicacid (either alone or with additional factors including otherrecombinase catalytic domains which may or may not form part of thefusion protein). In some embodiments, a catalytic domain of arecombinase does not include the DNA binding domain of the recombinase.In some embodiments, the catalytic domain of a recombinase includes partor all of a recombinase, e.g., the catalytic domain may include arecombinase domain and a DNA binding domain, or parts thereof, or thecatalytic domain may include a recombinase domain and a DNA bindingdomain that is mutated or truncated to abolish DNA binding activity.Recombinases and catalytic domains of recombinases are known to those ofskill in the art, and include, for example, those described herein. Insome embodiments, the catalytic domain is derived from any recombinase.In some embodiments, the recombinase catalytic domain is a catalyticdomain of aTn3 resolvase, a Hin recombinase, or a Gin recombinase. Insome embodiments, the catalytic domain comprises a Tn3 resolvase (e.g.,Stark Tn3 recombinase) that is encoded by a nucleotide sequencecomprising, in part or in whole, SEQ ID NO:55, as provided below. Insome embodiments, a Tn3 catalytic domain is encoded by a variant of SEQID NO:55. In some embodiments, a Tn3 catalytic domain is encoded by apolynucleotide (or a variant thereof) that encodes the polypeptidecorresponding to SEQ ID NO:56. In some embodiments, the catalytic domaincomprises a Hin recombinase that is encoded by a nucleotide sequencecomprising, in part or in whole, SEQ ID NO:57, as provided below. Insome embodiments, a Hin catalytic domain is encoded by a variant of SEQID NO:57. In some embodiments, a Hin catalytic domain is encoded by apolynucleotide (or a variant thereof) that encodes the polypeptidecorresponding to SEQ ID NO:58. In some embodiments, the catalytic domaincomprises a Gin recombinase (e.g., Gin beta recombinase) that is encodedby a nucleotide sequence comprising, in part or in whole, SEQ ID NO:59,as provided below. In some embodiments, a Gin catalytic domain isencoded by a variant of SEQ ID NO:59. In some embodiments, a Gincatalytic domain is encoded by a polynucleotide (or a variant thereof)that encodes the polypeptide corresponding to SEQ ID NO:60. Otherexemplary compositions and methods of using dCas9-recombinase fusionproteins can be found in U.S. patent application Ser. No. 14/320,467;titled “Cas9 Variants and Uses Thereof,” filed Jun. 30, 2014; the entirecontents of which are incorporated herein by reference.

Stark Tn3 recombinase (nucleotide: SEQ ID NO: 55;amino acid: SEQ ID NO: 56): (SEQ ID NO: 55)ATGGCCCTGTTTGGCTACGCACGCGTGTCTACCAGTCAACAGTCACTCGATTTGCAAGTGAGGGCTCTTAAAGATGCCGGAGTGAAGGCAAACAGAATTTTTACTGATAAGGCCAGCGGAAGCAGCACAGACAGAGAGGGGCTGGATCTCCTGAGAATGAAGGTAAAGGAGGGTGATGTGATCTTGGTCAAAAAATTGGATCGACTGGGGAGAGACACAGCTGATATGCTTCAGCTTATTAAAGAGTTTGACGCTCAGGGTGTTGCCGTGAGGTTTATCGATGACGGCATCTCAACCGACTCCTACATTGGTCTTATGTTTGTGACAATTTTGTCCGCTGTGGCTCAGGCTGAGCGGAGAAGGATTCTCGAAAGGACGAATGAGGGACGGCAAGCAGCTAAGTTGAAAGGTATCAAATTTGGCAGACGAAGG (SEQ ID NO: 56)MALFGYARVSTSQQSLDLQVRALKDAGVKANRIFTDKASGSSTDREGLDLLRMKVKEGDVILVKKLDRLGRDTADMLQLIKEFDAQGVAVRFIDDGISTDSYIGLMFVTILSAVAQAERRRILERTNEGRQAAKLKGIKFGRRRHin Recombinase (nucleotide: SEQ ID NO: 57; amino acid: SEQ ID NO: 58):(SEQ ID NO: 57) ATGGCAACCATTGGCTACATAAGGGTGTCTACCATCGACCAAAATATCGACCTGCAGCGCAACGCTCTGACATCCGCCAACTGCGATCGGATCTTCGAGGATAGGATCAGTGGCAAGATCGCCAACCGGCCCGGTCTGAAGCGGGCTCTGAAGTACGTGAATAAGGGCGATACTCTGGTTGTGTGGAAGTTGGATCGCTTGGGTAGATCAGTGAAGAATCTCGTAGCCCTGATAAGCGAGCTGCACGAGAGGGGTGCACATTTCCATTCTCTGACCGATTCCATCGATACGTCTAGCGCCATGGGCCGATTCTTCTTTTACGTCATGTCCGCCCTCGCTGAAATGGAGCGCGAACTTATTGTTGAACGGACTTTGGCTGGACTGGCAGCGGCTAGAGCAC AGGGCCGACTTGGA(SEQ ID NO: 58) MATIGYIRVSTIDQNIDLQRNALTSANCDRIFEDRISGKIANRPGLKRALKYVNKGDTLVVWKLDRLGRSVKNLVALISELHERGAHFHSLTDSIDTSSAMGRFFFYVMSALAEMERELIVERTLAGLAAARAQGRLGGin beta recombinase (nucleotide: SEQ ID NO: 59;amino acid: SEQ ID NO: 60): (SEQ ID NO: 59)ATGCTCATTGGCTATGTAAGGGTCAGCACCAATGACCAAAACACAGACTTGCAACGCAATGCTTTGGTTTGCGCCGGATGTGAACAGATATTTGAAGATAAACTGAGCGGCACTCGGACAGACAGACCTGGGCTTAAGAGAGCACTGAAAAGACTGCAGAAGGGGGACACCCTGGTCGTCTGGAAACTGGATCGCCTCGGACGCAGCATGAAACATCTGATTAGCCTGGTTGGTGAGCTTAGGGAGAGAGGAATCAACTTCAGAAGCCTGACCGACTCCATCGACACCAGTAGCCCCATGGGACGATTCTTCTTCTATGTGATGGGAGCACTTGCTGAGATGGAAAGAGAGCTTATTATCGAAAGAACTATGGCTGGTATCGCTGCTGCCCGGAACAAAGGCAGACGGTTCGGCAGACCGCCGAAGAGCGGC (SEQ ID NO: 60)MLIGYVRVSTNDQNTDLQRNALVCAGCEQIFEDKLSGTRTDRPGLKRALKRLQKGDTLVVWKLDRLGRSMKHLISLVGELRERGINFRSLTDSIDTSSPMGRFFFYVMGALAEMERELIIERTMAGIAAARNKGRRFGRPPKSG

In some embodiments, the enzymatic domain comprises a deaminase or acatalytic domain thereof. For example, in some embodiments, the generalarchitecture of exemplary dCas9 fusion proteins with a deaminase enzymeor domain comprises the structure:[NH₂]-[NLS]-Cas9-[deaminase]-[COOH],[NH₂]-[NLS]-[deaminase]-[Cas9]-[COOH],[NH₂]-[Cas9]-[deaminase]-[COOH], or[NH₂]-[deaminase]-[Cas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, a linker is inserted between the dCas9 and thedeaminase domain. In some embodiments, a linker is inserted between theNLS and the deaminase and/or dCas9 domain. In some embodiments, the NLSis located C-terminal of the deaminase and/or the dCas9 domain. In someembodiments, the NLS is located between the deaminase domain and thedCas9 domain. Additional features, such as sequence tags, may also bepresent. One exemplary suitable type of nucleic acid-editing enzymes anddomains are cytosine deaminases, for example, of the apolipoprotein BmRNA-editing complex (APOBEC) family of cytosine deaminase enzymes,including activation-induced cytidine deaminase (AID) and apolipoproteinB editing complex 3 (APOBEC3) enzyme. Another exemplary suitable type ofnucleic acid-editing enzyme and domain thereof suitable for use in thepresent invention include adenosine deaminases. For example, an ADATfamily adenosine deaminase can be fused to a dCas9 domain. Someexemplary suitable nucleic-acid editing enzymes and domains, e.g.,deaminases and deaminase domains, that can be fused to dCas9 domainsaccording to aspects of this disclosure are provided below. It will beunderstood that, in some embodiments, the active domain of therespective sequence can be used, e.g., the domain without a localizingsignal (nuclear localizing signal, without nuclear export signal,cytoplasmic localizing signal). Other exemplary compositions and methodsof using dCas9-nuclease fusion proteins can be found in U.S. patentapplication Ser. No. 14/325,815; titled “Fusions of Cas9 Domains andNucleic Acid-Editing Domains,” filed Jul. 8, 2014; the entire contentsof which are incorporated herein by reference.

Human AID: (SEQ ID NO: 61)MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization signal; double underline: nuclear export signal)Mouse AID: (SEQ ID NO: 62)MDSLLMKQKKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSCSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVAEFLRWNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIGIMTFKDYFYCWNTFVENRERTFKAWEGLHENSVRLTRQLRRILLPLYEVDDLRDAFRMLGF (underline: nuclear localization signal; double underline: nuclear export signal)Dog AID: (SEQ ID NO: 63)MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL(underline: nuclear localization signal; double underline: nuclear export signal)Bovine AID: (SEQ ID NO: 64)MDSLLKKQRQFLYQFKNVRWAKGRHETYLCYVVKRRDSPTSFSLDFGHLRNKAGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFTARLYFCDKERKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEVDDLRDAFRTLGL (underline: nuclear localization signal; double underline: nuclear export signal)Mouse APOBEC-3: (SEQ ID NO: 65)MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVTITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS(underline: nucleic acid editing domain) Rat APOBEC-3: (SEQ ID NO: 66)MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNIHAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFECAEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQHAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS(underline: nucleic acid editing domain) Rhesus macaque APOBEC-3G:(SEQ ID NO: 67)MVEPMDPRTFVSNFNNRPILSGLNTVWLCCEVKTKDPSGPPLDAKIFQGKVYSKAKY

ANSVATFLAKDPKVTLTIFVARLYYFWKPDYQQALRILCQKRGGPHATMKIMNYNEFQDCWNKFVDGRGKPFKPRNNLPKHYTLLQATLGELLRHLMDPGTFTSNFNNKPWVSGQHETYLCYKVERLHNDTWVPLNQHRGFLRNQAPNIHGFPKGR

AQEMAKFISNNEHVSLCIFAARIYDDQGRYQEGLRALHRDGAKIAMMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI(bold italic: nucleic acid editing domain; underline: cytoplasmic localizationsignal) Chimpanzee APOBEC-3G: (SEQ ID NO: 68)MKPHFRNPVERMYQDTFSDNFYNRPILSHRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSKLKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDVATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTSNFNNELWVRGRHETYLCYEVERLHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLHQDYRVTCFTSWSPCFSCAQEMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLAKAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLEEHSQALSGRLRAILQNQGN(underline: nucleic acid editing domain; double underline: cytoplasmiclocalization signal) Green monkey APOBEC-3G: (SEQ ID NO: 69)MNPQIRNMVEQMEPDIFVYYFNNRPILSGRNTVWLCYEVKTKDPSGPPLDANIFQGKLYPEAKDHPEMKFLHWFRKWRQLHRDQEYEVTWYVSWSPCTRCANSVATFLAEDPKVTLTIFVARLYYFWKPDYQQALRILCQERGGPHATMKIMNYNEFQHCWNEFVDGQGKPFKPRKNLPKHYTLLHATLGELLRHVMDPGTFTSNFNNKPWVSGQRETYLCYKVERSHNDTWVLLNQHRGFLRNQAPDRHGFPKGRHAELCFLDLIPFWKLDDQQYRVTCFTSWSPCFSCAQKMAKFISNNKHVSLCIFAARIYDDQGRCQEGLRTLHRDGAKIAVMNYSEFEYCWDTFVDRQGRPFQPWDGLDEHSQALSGRLRAI(underline: nucleic acid editing domain; double underline: cytoplasmiclocalization signal) Human APOBEC-3G: (SEQ ID NO: 70)MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPPLDAKIFRGQVYSELKYHPEMRFFHWFSKWRKLHRDQEYEVTWYISWSPCTKCTRDMATFLAEDPKVTLTIFVARLYYFWDPDYQEALRSLCQKRDGPRATMKIMNYDEFQHCWSKFVYSQRELFEPWNNLPKYYILLHIMLGEILRHSMDPPTFTFNFNNEPWVRGRHETYLCYEVERMHNDTWVLLNQRRGFLCNQAPHKHGFLEGRHAELCFLDVIPFWKLDLDQDYRVTCFTSWSPCFSCAQEMAKFISKNKHVSLCIFTARIYDDQGRCQEGLRTLAEAGAKISIMTYSEFKHCWDTFVDHQGCPFQPWDGLDEHSQDLSGRLRAILQNQEN(underline: nucleic acid editing domain; double underline: cytoplasmiclocalization signal) Human APOBEC-3F: (SEQ ID NO: 71)MKPHFRNTVERMYRDTFSYNFYNRPILSRRNTVWLCYEVKTKGPSRPRLDAKIFRGQVYSQPEHHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLAEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVKIMDDEEFAYCWENFVYSEGQPFMPWYKFDDNYAFLHRTLKEILRNPMEAMYPHIFYFHFKNLRKAYGRNESWLCFTMEVVKHHSPVSWKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLYYFWDTDYQEGLRSLSQEGASVEIMGYKDFKYCWENFVYNDDEPFKPWKGLKYNFLFLDSKLQEILE(underline: nucleic acid editing domain) Human APOBEC-3B:(SEQ ID NO: 72)MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQYHAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN(underline: nucleic acid editing domain) Human APOBEC-3C:(SEQ ID NO: 73)MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETHCHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDCAGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ(underline: nucleic acid editing domain) Human APOBEC-3A:(SEQ ID NO: 74)MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN(underline: nucleic acid editing domain) Human APOBEC-3H:(SEQ ID NO: 75)MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKCHAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSCAWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV(underline: nucleic acid editing domain) Human APOBEC-3D:(SEQ ID NO: 76)MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFENHAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPCVVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETHCHAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPECAGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (underline: nucleic acid editing domain) Human APOBEC-1:(SEQ ID NO: 77)MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR Mouse APOBEC-1: (SEQ ID NO: 78)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLKRat APOBEC-1: (SEQ ID NO: 79)MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKHuman ADAT-2: (SEQ ID NO: 80)MEAKAAPKPAASGACSVSAEETEKWMEEAMHMAKEALENTEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCRQSGKSPSEVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVEMLKTFYKQENPNAPKSKVRKKECQKS Mouse ADAT-2: (SEQ ID NO: 81)MEEKVESTTTPDGPCVVSVQETEKWMEEAMRMAKEALENIEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCHQHGQSPSTVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVELLKTFYKQENPNAPKSKVRKKDCQKS Mouse ADAT-1: (SEQ ID NO: 82)MWTADEIAQLCYAHYNVRLPKQGKPEPNREWTLLAAVVKIQASANQACDIPEKEVQVTKEVVSMGTGTKCIGQSKMRESGDILNDSHAEIIARRSFQRYLLHQLHLAAVLKEDSIFVPGTQRGLWRLRPDLSFVFFSSHTPCGDASIIPMLEFEEQPCCPVIRSWANNSPVQETENLEDSKDKRNCEDPASPVAKKMRLGTPARSLSNCVAHHGTQESGPVKPDVSSSDLTKEEPDAANGIASGSFRVVDVYRTGAKCVPGETGDLREPGAAYHQVGLLRVKPGRGDRTCSMSCSDKMARWNVLGCQGALLMHFLEKPIYLSAVVIGKCPYSQEAMRRALTGRCEETLVLPRGFGVQELEIQQSGLLFEQSRCAVHRKRGDSPGRLVPCGAAISWSAVPQQPLDVTANGFPQGTTKKEIGSPRARSRISKVELFRSFQKLLSSIADDEQPDSIRVTKKLDTYQEYKDAASAYQEAWGALRRIQPFASWIRNPPDYHQFK  (underline: nucleic acid editingdomain) Human ADAT-1: (SEQ ID NO: 83)MWTADEIAQLCYEHYGIRLPKKGKPEPNHEWTLLAAVVKIQSPADKACDTPDKPVQVTKEVVSMGTGTKCIGQSKMRKNGDILNDSHAEVIARRSFQRYLLHQLQLAATLKEDSIFVPGTQKGVWKLRRDLIFVFFSSHTPCGDASIIPMLEFEDQPCCPVFRNWAHNSSVEASSNLEAPGNERKCEDPDSPVTKKMRLEPGTAAREVTNGAAHHQSFGKQKSGPISPGIHSCDLTVEGLATVTRIAPGSAKVIDVYRTGAKCVPGEAGDSGKPGAAFHQVGLLRVKPGRGDRTRSMSCSDKMARWNVLGCQGALLMHLLEEPIYLSAVVIGKCPYSQEAMQRALIGRCQNVSALPKGFGVQELKILQSDLLFEQSRSAVQAKRADSPGRLVPCGAAISWSAVPEQPLDVTANGFPQGTTKKTIGSLQARSQISKVELFRSFQKLLSRIARDKWPHSLRVQKLDTYQEYKEAASYQEAWSTLRKQVFGSWIRNPPDYHQFK  (underline: nucleic acidediting domain)

In some embodiments, the enzymatic domain comprises one or more of atranscriptional activator. For example, in some embodiments, the generalarchitecture of exemplary dCas9 fusion proteins with a transcriptionalactivator domain comprises the structure:[NH₂]-[NLS]-[Cas9]-[(transcriptional activator)_(n)]-[COOH],[NH₂]-[NLS]-[(transcriptional activator)_(n)]-[Cas9]-1-[COOH],[NH₂]-[Cas9]-[(transcriptional activator)_(n)]-[COOH], or[NH₂]-[(transcriptional activator)_(n)]-[Cas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, the fusion proteins comprises one or more repeats ofthe transcriptional activator, for example wherein n=1-10 (e.g., n is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n=1-20. In someembodiments, a linker is inserted between the dCas9 and thetranscriptional activator domain. In some embodiments, a linker isinserted between the NLS and the transcriptional activator and/or dCas9domain. In some embodiments, the NLS is located C-terminal of thetranscriptional activator and/or the dCas9 domain. In some embodiments,the NLS is located between the transcriptional activator domain and thedCas9 domain. Additional features, such as sequence tags, may also bepresent. In some embodiments, the transcriptional activator is selectedfrom the group consisting of VP64, (SEQ ID NO:84 or SEQ ID NO:35), VP16(SEQ ID NO:85), and p65 (SEQ ID NO:86). In some embodiments, adCas9-VP64 fusion protein comprises the amino acid sequence of SEQ IDNO:87 (e.g., with or without the 6×His tag) or comprises an amino acidsequence that is at least 80%, at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identical to the amino acid sequence ofSEQ ID NO:87 (e.g., with or without the 6×His tag).

VP64 (SEQ ID NO: 84) GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN VP16 (SEQ ID NO: 85)APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGEFPGIRR p65: (SEQ ID NO: 86)PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQ dCas9-VP64-6xHis: (SEQ ID NO: 87)MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSSDYKDHDGDYKDHDIDYKDDDDKAAGGGGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS DALDDFDLDMLHHHHHH

In some embodiments, the enzymatic domain comprises one or more of atranscriptional repressor. For example, in some embodiments, the generalarchitecture of exemplary dCas9 fusion proteins with a transcriptionalrepressor domain comprises the structure:[NH₂]-[NLS]-[Cas9]-[(transcriptional repressor)_(n)]-[COOH],[NH₂]-[NLS]-[(transcriptional repressor)_(n)]-[Cas9]-[COOH],[NH₂]-[Cas9]-[(transcriptional repressor)_(n)]-[COOH], or[NH₂]-[(transcriptional repressor)_(n)]-[Cas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, the fusion proteins comprises one or more repeats ofthe transcriptional repressor, for example wherein n=1-10 (e.g., n is 1,2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n=1-20. In someembodiments, a linker is inserted between the dCas9 and thetranscriptional repressor domain. In some embodiments, a linker isinserted between the NLS and the transcriptional repressor and/or dCas9domain. In some embodiments, the NLS is located C-terminal of thetranscriptional repressor and/or the dCas9 domain. In some embodiments,the NLS is located between the transcriptional repressor domain and thedCas9 domain. Additional features, such as sequence tags, may also bepresent. In some embodiments, the transcriptional repressor is selectedfrom the group consisting of the KRAB (Kruppel associated box) domain ofKox1, SID (mSin3 interaction domain), the CS (Chromo Shadow) domain ofHP1α, or the WRPW domain of Hesl. These and other repressor domains areknown in the art, and in some embodiments correspond to those describedin Urrutia, KRAB-containing zinc-finger repressor proteins. Genome Biol.2003; 4(10):231; Gilbert et al. CRISPR-mediated modular RNA-guidedregulation of transcription in eukaryotes. Cell. 2013; 154, 442-451;Konermann et al., Optical control of mammalian endogenous transcriptionand epigenetic states. Nature. 2013; 500, 472-476; and published U.S.patent application Ser. No. 14/105,017, published as U.S. 2014/0186958A1, the entire contents of which are incorporated herein by reference.In some embodiments, the transcription repressor domain comprises one ormore repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats) of a KRABdomain. In some embodiments, the KRAB domain comprises an amino acidsequence selected from the group consisting of SEQ ID NOs:88-91. In someembodiments, the transcriptional repressor domains comprises one or morerepeats of a SID protein. In some embodiments, the SID protein comprisesan amino acid sequence set forth as SEQ ID NO:80. In some embodiments,the repressor domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats ofa SID protein (e.g., SEQ ID NO:92). In some embodiments, the repressordomain comprises four repeats of SID (e.g., SID4x; SEQ ID NO:93).

KRAB (human; GenBank: AAD20972.1) (SEQ ID NO: 88)MNMFKEAVTFKDVAVAFTEEELGLLGPAQRKLYRDVMVENFRNLLSVGHPPFKQDVSPIERNEQLWIMTTATRRQGNLDTLPVKALLLYDLAQTKRAB protein domain, partial (human; GenBank: CAB52478.1):(SEQ ID NO: 89) EQVSFKDVCVDFTQEEWYLLDPAQKILYRDVILENYSNLVSVGYCITKPEVIFKIEQGEEPWILEKGFPSQCHP KRAB A domain, partial (human; GenBank:AAB03530.1): (SEQ ID NO: 90) EAVTFKDVAVVFTEEELGLLDPAQRKLYRDVMLENFRNLLSVKRAB (mouse; C2H2 type domain containing protein; GenBank: CAM27971.1):(SEQ ID NO: 91) MDLVTYDDVHVNFTQDEWALLDPSQKSLYKGVMLETYKNLTAIGYIWEEHTIEDHFQTSRSHGSNKKTH SID repressor domain: (SEQ ID NO: 92)GSGMNIQMLLEAADYLERREREAEHGYASMLP SID4x repressor domain: (SEQ ID NO: 93)GSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPSR

In some embodiments, the enzymatic domain comprises an epigeneticmodifier or a catalytic domain thereof. For example, in someembodiments, the general architecture of exemplary dCas9 fusion proteinswith an epigenetic modifier or domain comprises the structure:[NH₂]-[NLS]-[Cas9]-[epigenetic modifier]-[COOH],[NH₂]-[NLS]-[epigenetic modifier]-[Cas9]-[COOH],[NH₂]-[Cas9]-[epigenetic modifier]-[COOH], or[NH₂]-[epigenetic modifier]-[Cas9]-[COOH];wherein NLS is a nuclear localization signal, NH₂ is the N-terminus ofthe fusion protein, and COOH is the C-terminus of the fusion protein. Insome embodiments, a linker is inserted between the dCas9 and theepigenetic modifier domain. In some embodiments, a linker is insertedbetween the NLS and the epigenetic modifier and/or dCas9 domain. In someembodiments, the NLS is located C-terminal of the epigenetic modifierand/or the dCas9 domain. In some embodiments, the NLS is located betweenthe epigenetic modifier domain and the dCas9 domain. Additionalfeatures, such as sequence tags, may also be present. Epigeneticmodifiers are well known in the art, and typically catalyze DNAmethylation (and demethylation) or histone modifications (e.g., histonemethylation/demethylation, acetylation/deacetylation, ubiquitylation,phosphorylation, sumoylation, etc.). The presence of one more epigeneticmodifications can affect the transcriptional activity of one or moregenes, for example turning genes from an “on” state to an “off” state,and vice versa. Epigenetic modifiers include, but are not limited to,histone demethylase, histone methyltransferase, hydroxylase, histonedeacetylase, and histone acetyltransferase. Exemplary epigeneticmodifying proteins can be found in Konermann et al., Optical control ofmammalian endogenous transcription and epigenetic states. Nature. 2013;500, 472-476; Mendenhall et al., Locus-specific editing of histonemodifications at endogenous enhancers. Nat. Biotechnol. 2013; 31,1133-1136; and Maeder et al., Targeted DNA demethylation and activationof endogenous genes using programmable TALE-TET1 fusion proteins. Nat.Biotechnol. 2013; 31, 1137-1142; the entire contents of each areincorporated herein by reference. In some embodiments, the epigeneticmodifier domain is LSD1 (Lysine (K)-specific demethylase 1A) histonedemethylase, which in some embodiments, comprises in whole or in part,an amino acid sequence set forth as SEQ ID NO:94 or SEQ ID NO:95. Insome embodiments, the epigenetic modifier domain is TET1 hydroxylasecatalytic domain, which in some embodiments, comprises an amino acidsequence set forth as SEQ ID NO:96. In some embodiments, the epigeneticmodifier is a histone deacetylase (HDAC) effector domain. In someembodiments, the HDAC effector domain comprises in whole in in part, anamino acid sequence corresponding to any of the HDAC effector proteinsprovided in Supplementary Table 2 of Konermann et al., Optical controlof mammalian endogenous transcription and epigenetic states. Nature.2013; 500, 472-476; SEQ ID NOs:97-108. In some embodiments, theepigenetic modifier is a histone methyltransferase (HMT) effectordomain. In some embodiments, the HMT effector domain comprises in wholein in part, an amino acid sequence corresponding to any of the HDACeffector proteins provided in Supplementary Table 3 of Konermann et al.,Optical control of mammalian endogenous transcription and epigeneticstates. Nature. 2013; 500, 472-476; SEQ ID NOs:109-118.

LSD1, isoform a (human): (SEQ ID NO: 94)MLSGKKAAAAAAAAAAAATGTEAGPGTAGGSENGSEVAAQPAGLSGPAEVGPGAVGERTPRKKEPPRASPPGGLAEPPGSAGPQAGPTVVPGSATPMETGIAETPEGRRTSRRKRAKVEYREMDESLANLSEDEYYSEEERNAKAEKEKKLPPPPPQAPPEEENESEPEEPSGQAGGLQDDSSGGYGDGQASGVEGAAFQSRLPHDRMTSQEAACFPDIISGPQQTQKVFLFIRNRTLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLERHGLINFGIYKRIKPLPTKKTGKVIIIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFRKGNYVADLGAMVVTGLGGNPMAVVSKQVNMELAKIKQKCPLYEANGQADTVKVPKEKDEMVEQEFNRLLEATSYLSHQLDFNVLNNKPVSLGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMVNLKEKIKELHQQYKEASEVKPPRDITAEFLVKSKHRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLSSRDRQILDWHFANLEFANATPLSTLSLKHWDQDDDFEFTGSHLTVRNGYSCVPVALAEGLDIKLNTAVRQVRYTASGCEVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVLCFDRVFWDPSVNLFGHVGSTTASRGELFLFWNLYKAPILLALVAGEAAGIMENISDDVIVGRCLAILKGIFGSSAVPQPKETVVSRWRADPWARGSYSYVAAGSSGNDYDLMAQPITPGPSIPGAPQPIPRLFFAGEHTIRNYPATVHGALLSGLREAGRIADQFLGAMYTLPRQATPGVPAQQSPSM LSD1, isoform b (human):(SEQ ID NO: 95)MLSGKKAAAAAAAAAAAATGTEAGPGTAGGSENGSEVAAQPAGLSGPAEVGPGAVGERTPRKKEPPRASPPGGLAEPPGSAGPQAGPTVVPGSATPMETGIAETPEGRRTSRRKRAKVEYREMDESLANLSEDEYYSEEERNAKAEKEKKLPPPPPQAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTSQEAACFPDIISGPQQTQKVFLFIRNRTLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLERHGLINFGIYKRIKPLPTKKTGKVIIIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFRKGNYVADLGAMVVTGLGGNPMAVVSKQVNMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRLLEATSYLSHQLDFNVLNNKPVSLGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMVNLKEKIKELHQQYKEASEVKPPRDITAEFLVKSKHRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLSSRDRQILDWHFANLEFANATPLSTLSLKHWDQDDDFEFTGSHLTVRNGYSCVPVALAEGLDIKLNTAVRQVRYTASGCEVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVLCFDRVFWDPSVNLFGHVGSTTASRGELFLFWNLYKAPILLALVAGEAAGIMENISDDVIVGRCLAILKGIFGSSAVPQPKETVVSRWRADPWARGSYSYVAAGSSGNDYDLMAQPITPGPSIPGAPQPIPRLFFAGEHTIRNYPATVHGALLSGLREAGRIADQFLGAMYTLPRQATPGVPAQQSPSM TET1 catalytic domain: (SEQ ID NO: 96)SIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGSLPTCSCLDRVIQKDKGPYYTHLGAGPSVAAVREIMENRYGQKGNAIRIEIVVYTGKEGKSSHGCPIAKWVLRRSSDEEKVLCLVRQRTGHHCPTAVMVVLIMVWDGIPLPMADRLYTELTENLKSYNGHPTDRRCTLNENRTCTCQGIDPETCGASFSFGCSWSMYFNGCKFGRSPSPRRFRIDPSSPLHEKNLEDNLQSLATRLAPIYKQYAPVAYQNQVEYENVARECRLGSKEGRPFSGVTACLDFCAHPHRDIHNMNNGSTVVCTLTREDNRSLGVIPQDEQLHVLPLYKLSDTDEFGSKEGMEAKIKSGAIEVLAPRRKKRTCFTQPVPRSGKKRAAMMTEVLAHKIRAVEKKPIPRIKRKNNSTTTNNSKPSSLPTLGSNTETVQPEVKSETEPHFILKSSDNTKTYSLMPSAPHPVKEASPGFSWSPKTASATPAPLKNDATASCGFSERSSTPHCTMPSGRLSGANAAAADGPGISQLGEVAPLPTLSAPVMEPLINSEPSTGVTEPLTPHQPNHQPSFLTSPQDLASSPMEEDEQHSEADEPPSDEPLSDDPLSPAEEKLPHIDEYWSDSEHIFLDANIGGVAIAPAHGSVLIECARRELHATTPVEHPNRNHPTRLSLVFYQHKNLNKPQHGFELNKIKFEAKEAKNKKMKASEQKDQAANEGPEQSSEVNELNQIPSHKALTLTHDNVVTVSPYALTHVAGPYNHWV HDAC effector domains:HDAC8 (X. laevis): (SEQ ID NO: 97)ASSPKKKRKVEASMSRVVKPKVASMEEMAAFHTDAYLQHLHKVSEEGDNDDPETLEYGLGYDCPITEGIYDYAAAVGGATLTAAEQLIEGKTRIAVNWPGGWHHAKKDEASGFCYLNDAVLGILKLREKFDRVLYVDMDLHHGDGVEDAFSFTSKVMTVSLHKFSPGFFPGTGDVSDIGLGKGRYYSINVPLQDGIQDDKYYQICEGVLKEVFTTFNPEAVVLQLGADTIAGDPMCSFNMTPEGIGKCLKYVLQWQLPTLILGGGGYHLPNTARCWTYLTALIVGRTLSSEIPDHEFFTEYGPDYVLEITPSCRPDRNDTQKVQEILQSIKGNLKRVVEFRPD3 (S. cerevisiae): (SEQ ID NO: 98)ASSPKKKRKVEASRRVAYFYDADVGNYAYGAGHPMKPHRIRMAHSLIMNYGLYKKMEIYRAKPATKQEMCQFHTDEYIDFLSRVTPDNLEMFKRESVKFNVGDDCPVFDGLYEYCSISGGGSMEGAARLNRGKCDVAVNYAGGLHHAKKSEASGFCYLNDIVLGIIELLRYHPRVLYIDIDVHHGDGVEEAFYTTDRVMTCSFHKYGEFFPGTGELRDIGVGAGKNYAVNVPLRDGIDDATYRSVFEPVIKKIMEWYQPSAVVLQCGGDSLSGDRLGCFNLSMEGHANCVNYVKSFGIPMMVVGGGGYTMRNVARTWCFETGLLNNVVLDKDLPYEFMesoLo4 (M. loti): (SEQ ID NO: 99)ASSPKKKRKVEASMPLQIVHHPDYDAGFATNHRFPMSKYPLLMEALRARGLASPDALNTTEPAPASWLKLAHAADYVDQVISCSVPEKIEREIGFPVGPRVSLRAQLATGGTILAARLALRHGIACNTAGGSHHARRAQGAGFCTFNDVAVASLVLLDEGAAQNILVVDLDVHQGDGTADILSDEPGVFTFSMHGERNYPVRKIASDLDIALPDGTGDAAYLRRLATILPELSARARWDIVFYNAGVDVHAEDRLGRLALSNGGLRARDEMVIGHFRALGIPVCGVIGGGYSTDVPALASRHAILFEVASTYAEF HDAC11 (human): (SEQ ID NO: 100)ASSPKKKRKVEASMLHTTQLYQHVPETRWPIVYSPRYNITFMGLEKLHPFDAGKWGKVINFLKEEKLLSDSMLVEAREASEEDLLVVHTRRYLNELKWSFAVATITEIPPVIFLPNFLVQRKVLRPLRTQTGGTIMAGKLAVERGWAINVGGGFHHCSSDRGGGFCAYADITLAIKFLFERVEGISRATIIDLDAHQGNGHERDFMDDKRVYIMDVYNRHIYPGDRFAKQAIRRKVELEWGTEDDEYLDKVERNIKKSLQEHLPDVVVYNAGTDILEGDRLGGLSISPAGIVKRDELVFRMVRGRRVPILMVTSGGYQKRTARIIADSILNLFGLGLIGPESPSVSAQNSDTPLLPPAVPEF HDT1 (A. thaliana): (SEQ ID NO: 101)ASSPKKKRKVEASMEFWGIEVKSGKPVTVTPEEGILIHVSQASLGECKNKKGEFVPLHVKVGNQNLVLGTLSTENIPQLFCDLVFDKEFELSHTWGKGSVYFVGYKTPNIEPQGYSEEEEEEEEEVPAGNAAKAVAKPKAKPAEVKPAVDDEEDESDSDGMDEDDSDGEDSEEEEPTPKKPASSKKRANETTPKAPVSAKKAKVAVTPQKTDEKKKGGKAANQSEF SIRT3 (human): (SEQ ID NO: 102)ASSPKKKRKVEASMVGAGISTPSGIPDFRSPGSGLYSNLQQYDLPYPEAIFELPFFFHNPKPFFTLAKELYPGNYKPNVTHYFLRLLHDKGLLLRLYTQNIDGLERVSGIPASKLVEAHGTFASATCTVCQRPFPGEDIRADVMADRVPRCPVCTGVVKPDIVFFGEPLPQRFLLHVVDFPMADLLLILGTSLEVEPFASLTEAVRSSVPRLLINRDLVGPLAWHPRSRDVAQLGDVVHGVESLVELLGWTEEMRDLVQRETGKLDGPDKEFHST2 (S. cerevisiae): (SEQ ID NO: 103)ASSPKKKRKVEASTEMSVRKIAAHMKSNPNAKVIFMVGAGISTSCGIPDFRSPGTGLYHNLARLKLPYPEAVFDVDFFQSDPLPFYTLAKELYPGNFRPSKFHYLLKLFQDKDVLKRVYTQNIDTLERQAGVKDDLIIEAHGSFAHCHCIGCGKVYPPQVFKSKLAEHPIKDFVKCDVCGELVKPAIVFFGEDLPDSFSETWLNDSEWLREKITTSGKHPQQPLVIVVGTSLAVYPFASLPEEIPRKVKRVLCNLETVGDFKANKRPTDLIVHQYSDEFAEQLVEELGWQEDFEKILTAQGGMGEF CobB (E. coli (K12)): (SEQ ID NO: 104)ASSPKKKRKVEASMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMPLGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAEF HST2 (C. albicans):(SEQ ID NO: 105)ASSPKKKRKVEASMPSLDDILKPVAEAVKNGKKVTFFNGAGISTGAGIPDFRSPDTGLYANLAKLNLPFAEAVFDIDFFKEDPKPFYTLAEELYPGNFAPTKFHHFIKLLQDQGSLKRVYTQNIDTLERLAGVEDKYIVEAHGSFASNHCVDCHKEMTTETLKTYMKDKKIPSCQHCEGYVKPDIVFFGEGLPVKFFDLWEDDCEDVEVAIVAGTSLTVFPFASLPGEVNKKCLRVLVNKEKVGTFKHEPRKSDIIALHDCDIVAERLCTLLGLDDKLNEVYEKEKIKYSKAETKEIKMHEIEDKLKEEAHLKEDKHTTKVDKKEKQNDANDKELEQLIDKAKAEFSIRT5 (human): (SEQ ID NO: 106)ASSPKKKRKVEASSSSMADFRKFFAKAKHIVIISGAGVSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELAHCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEALACHENETVSEF Sir2A (P. falciparum): (SEQ ID NO: 107)ASSPKKKRKVEASMGNLMISFLKKDTQSITLEELAKIIKKCKHVVALTGSGTSAESNIPSFRGSSNSIWSKYDPRIYGTIWGFWKYPEKIWEVIRDISSDYEIEINNGHVALSTLESLGYLKSVVTQNVDGLHEASGNTKVISLHGNVFEAVCCTCNKIVKLNKIMLQKTSHFMHQLPPECPCGGIFKPNIILFGEVVSSDLLKEAEEEIAKCDLLLVIGTSSTVSTATNLCHFACKKKKKIVEINISKTYITNKMSDYHVCAKFSELTKVANILKGSSEKNKKIMEF SIRT6 (human): (SEQ ID NO: 108)ASSPKKKRKVEASMSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLEF HMT effector domains: NUE (C. trachomatis):(SEQ ID NO: 109)ASSPKKKRKVEASMTTNSTQDTLYLSLHGGIDSAIPYPVRRVEQLLQFSFLPELQFQNAAVKQRIQRLCYREEKRLAVSSLAKWLGQLHKQRLRAPKNPPVAICWINSYVGYGVFARESIPAWSYIGEYTGILRRRQALWLDENDYCFRYPVPRYSFRYFTIDSGMQGNVTRFINHSDNPNLEAIGAFENGIFHIIIRAIKDILPGEELCYHYGPLYWKHRKKREEFVPQEEEF vSET (P. bursaria chlorella virus):(SEQ ID NO: 110)ASSPKKKRKVEASMFNDRVIVKKSPLGGYGVFARKSFEKGELVEECLCIVRHNDDWGTALEDYLFSRKNMSAMALGFGAIFNHSKDPNARHELTAGLKRMRIFTIKPIAIGEEITISYGDDYWLSRPRLTQNEFSUV39H1 (human): (SEQ ID NO: 111)ASSPKKKRKVEASNLKCVRILKQFHKDLERELLRRHHRSKTPRHLDPSLANYLVQKAKQRRALRRWEQELNAKRSHLGRITVENEVDLDGPPRAFVYINEYRVGEGITLNQVAVGCECQDCLWAPTGGCCPGASLHKFAYNDQGQVRLRAGLPIYECNSRCRCGYDCPNRVVQKGIRYDLCIFRTDDGRGWGVRTLEKIRKNSFVMEYVGEIITSEEAERRGQIYDRQGATYLFDLDYVEDVYTVDAAYYGNISHFVNHSCDPNLQVYNVFIDNLDERLPRIAFFATRTIRAGEELTFDYNMQVDPVDMESTRMDSNFGLAGLPGSPKKRVRIECKCGTESCRKYLFEFDIM5 (N. crassa): (SEQ ID NO: 112)ASSPKKKRKVEASMEKAFRPHFFNHGKPDANPKEKKNCHWCQIRSFATHAQLPISIVNREDDAFLNPNFRFIDHSIIGKNVPVADQSFRVGCSCASDEECMYSTCQCLDEMAPDSDEEADPYTRKKRFAYYSQGAKKGLLRDRVLQSQEPIYECHQGCACSKDCPNRVVERGRTVPLQIFRTKDRGWGVKCPVNIKRGQFVDRYLGEIITSEEADRRRAESTIARRKDVYLFALDKFSDPDSLDPLLAGQPLEVDGEYMSGPTRFINHSCDPNMAIFARVGDHADKHIHDLALFAIKDIPKGTELTFDYVNGLTGLESDAHDPSKISEMTKCLCGTAKCRGYLWEFKYP (A. thaliana): (SEQ ID NO: 113)ASSPKKKRKVEASDISGGLEFKGIPATNRVDDSPVSPTSGFTYIKSLIIEPNVIIPKSSTGCNCRGSCTDSKKCACAKLNGGNFPYVDLNDGRLIESRDVVFECGPHCGCGPKCVNRTSQKRLRFNLEVFRSAKKGWAVRSWEYIPAGSPVCEYIGVVRRTADVDTISDNEYIFEIDCQQTMQGLGGRQRRLRDVAVPMNNGVSQSSEDENAPEFCIDAGSTGNFARFINHSCEPNLFVQCVLSSHQDIRLARVVLFAADNISPMQELTYDYGYALDSVHEF SUVR4 (A. thaliana): (SEQ ID NO: 114)ASSPKKKRKVEASQSAYLHVSLARISDEDCCANCKGNCLSADFPCTCARETSGEYAYTKEGLLKEKFLDTCLKMKKEPDSFPKVYCKDCPLERDHDKGTYGKCDGHLIRKFIKECWRKCGCDMQCGNRVVQRGIRCQLQVYFTQEGKGWGLRTLQDLPKGTFICEYIGEILTNTELYDRNVRSSSERHTYPVTLDADWGSEKDLKDEEALCLDATICGNVARFINHRCEDANMIDIPIEIETPDRHYYHIAFFTLRDVKAMDELTWDYMIDFNDKSHPVKAFRCCCGSESCRDRKIKGSQGKSIERRKIVSAKKQQGSKEVSKKRKEF Set4 (C. elegans):(SEQ ID NO: 115)ASSPKKKRKVEASMQLHEQIANISVTFNDIPRSDHSMTPTELCYFDDFATTLVVDSVLNFTTHKMSKKRRYLYQDEYRTARTVMKTFREQRDWTNAIYGLLTLRSVSHFLSKLPPNKLFEFRDHIVRFLNMFILDSGYTIQECKRYSQEGHQGAKLVSTGVWSRGDKIERLSGVVCLLSSEDEDSILAQEGSDFSVMYSTRKRCSTLWLGPGAYINHDCRPTCEFVSHGSTAHIRVLRDMVPGDEITCFYGSEFFGPNNIDCECCTCEKNMNGAFSYLRGNENAEPIISEKKTKYELRSRSEF Set1 (C. elegans): (SEQ ID NO: 116)ASSPKKKRKVEASMKVAAKKLATSRMRKDRAAAASPSSDIENSENPSSLASHSSSSGRMTPSKNTRSRKGVSVKDVSNHKITEFFQVRRSNRKTSKQISDEAKHALRDTVLKGTNERLLEVYKDVVKGRGIRTKVNFEKGDFVVEYRGVMMEYSEAKVIEEQYSNDEEIGSYMYFFEHNNKKWCIDATKESPWKGRLINHSVLRPNLKTKVVEIDGSHHLILVARRQIAQGEELLYDYGDRSAETIAKNPWLVNTEF SETD8 (human)(SEQ ID NO: 117)ASSPKKKRKVEASSCDSTNAAIAKQALKKPIKGKQAPRKKAQGKTQQNRKLTDFYPVRRSSRKSKAELQSEERKRIDELIESGKEEGMKIDLIDGKGRGVIATKQFSRGDFVVEYHGDLIEITDAKKREALYAQDPSTGCYMYYFQYLSKTYCVDATRETNRLGRLINHSKCGNCQTKLHDIDGVPHLILIASRDIAAGEELLYDYGDRSKASIEAFPWLKHEF TgSET8 (T. gondii): (SEQ ID NO: 118)ASSPKKKRKVEASASRRTGEFLRDAQAPSRWLKRSKTGQDDGAFCLETWLAGAGDDAAGGERGRDREGAADKAKQREERRQKELEERFEEMKVEFEEKAQRMIARRAALTGEIYSDGKGSKKPRVPSLPENDDDALIEIIIDPEQGILKWPLSVMSIRQRTVIYQECLRRDLTACIHLTKVPGKGRAVFAADTILKDDFVVEYKGELCSEREAREREQRYNRSKVPMGSFMFYFKNGSRMMAIDATDEKQDFGPARLINHSRRNPNMTPRAITLGDFNSEPRLIFVARRNIEKGEELLVDYGERDPDVIKEHPWLNSEF

Those of skill in the art will understand that any of the exemplary Cas9proteins, including the exemplary Cas9 nucleases, variants, and fusionsthereof, e.g., described herein, can be delivered to cells using theinstantly disclosed technology, and that the disclosure is not limitedin this respect.

Nuclease Effector Proteins

TALE nucleases, or TALENs, are artificial nucleases comprising atranscriptional activator-like effector DNA binding domain associatedwith a DNA cleavage domain, for example, a FokI domain. A number ofmodular assembly schemes for generating engineered TALE constructs havebeen reported (Zhang, Feng; et. al. (February 2011). “Efficientconstruction of sequence-specific TAL effectors for modulating mammaliantranscription”. Nature Biotechnology 29 (2): 149-53; Geiβler, R.;Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, S. E.; Boch, J.(2011), Shiu, Shin-Han. ed. “Transcriptional Activators of Human Geneswith Programmable DNA-Specificity”. PLoS ONE 6 (5): e19509; Cermak, T.;Doyle, E. L.; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller,J. A.; Somia, N. V. et al. (2011). “Efficient design and assembly ofcustom TALEN and other TAL effector-based constructs for DNA targeting”.Nucleic Acids Research; Morbitzer, R.; Elsaesser, J.; Hausner, J.;Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains bymodular cloning”. Nucleic Acids Research; Li, T.; Huang, S.; Zhao, X.;Wright, D. A.; Carpenter, S.; Spalding, M. H.; Weeks, D. P.; Yang, B.(2011). “Modularly assembled designer TAL effector nucleases fortargeted gene knockout and gene replacement in eukaryotes”. NucleicAcids Research.; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.;Marillonnet, S. (2011). Bendahmane, Mohammed. ed. “Assembly of DesignerTAL Effectors by Golden Gate Cloning”. PLoS ONE 6 (5): e19722; theentire contents of each of which are incorporated herein by reference).Those of skill in the art will understand that TALE nucleases can beengineered to target virtually any genomic sequence with highspecificity, and that such engineered nucleases can be used inembodiments of the present technology to manipulate the genome of acell, e.g., by delivering the respective TALEN via a method or strategydisclosed herein under circumstances suitable for the TALEN to bind andcleave its target sequence within the genome of the cell. In someembodiments, the delivered TALEN targets a gene or allele associatedwith a disease or disorder. In some embodiments, delivery of the TALENto a subject confers a therapeutic benefit to the subject.

Zinc finger nucleases are a class of artificial nucleases that comprisea DNA cleavage domain and a zinc finger DNA binding domain. In someembodiments, the DNA cleavage domain is a non-specific DNA cleavagedomain of a restriction endonuclease, for example, of FokI. In someembodiments, the DNA cleavage domain is a domain that only cleavesdouble-stranded DNA when dimerized with a second DNA cleavage domain ofthe same type. In some embodiments, the DNA cleavage domain is fused tothe C-terminus of the zinc finger domain via a linker, for example, apeptide linker. In some embodiments, the zinc finger domain comprisesbetween about 3 and about 6 zinc fingers and specifically recognizes andbinds a target sequence of about 9-20 nucleotides in length. In someembodiments, a plurality of zinc finger nuclease molecules is deliveredto a target cell by a system or method provided by this invention, withthe zinc finger domain of one zinc finger nuclease molecule binding atarget sequence in close proximity of the target sequence of a secondzinc finger nuclease molecule. In some embodiments, the zinc fingerdomains of the zinc finger nuclease molecules binding target sequencesin close proximity to each other are different. In some embodiments, azinc finger nuclease molecule delivered to a cell by a system or methodprovided herein binds a target nucleic acid sequence in close proximityto the target sequence of another zinc finger nuclease molecule, so thatthe DNA cleavage domains of the molecules dimerize and cleave a DNAmolecule at a site between the two target sequences.

In some embodiments, the genome of the target cell is edited by anuclease delivered to the cell via a strategy or method disclosedherein, e.g., by a TALEN, or a zinc-finger nuclease, or a plurality orcombination of such nucleases. In some embodiments, a single- ordouble-strand break is introduced at a specific site within the genomeof a target cell by the nuclease, resulting in a disruption of thetargeted genomic sequence. In some embodiments, the targeted genomicsequence is a nucleic acid sequence within the coding region of a gene.In some embodiments, the strand break introduced by the nuclease leadsto a mutation within the target gene that impairs the expression of theencoded gene product. In some embodiments, a nucleic acid isco-delivered to the cell with the nuclease. In some embodiments, thenucleic acid comprises a sequence that is identical or homologous to asequence adjacent to the nuclease target site. In some such embodiments,the strand break effected by the nuclease is repaired by the cellularDNA repair machinery to introduce all or part of the co-deliverednucleic acid into the cellular DNA at the break site, resulting in atargeted insertion of the co-delivered nucleic acid, or part thereof. Insome embodiments, the insertion results in the disruption or repair of apathogenic allele. In some embodiments, the insertion is detected by asuitable assay, e.g., a DNA sequencing assay, a southern blot assay, oran assay for a reporter gene encoded by the co-delivered nucleic acid,e.g., a fluorescent protein or resistance to an antibiotic. In someembodiments, the nucleic acid is co-delivered by association to asupercharged protein. In some embodiments, the supercharged protein isalso associated to the functional effector protein, e.g., the nuclease.In some embodiments, the delivery of a nuclease to a target cell resultsin a clinically or therapeutically beneficial disruption of the functionof a gene.

In some embodiments, cells from a subject are obtained and a nuclease orother effector protein is delivered to the cells by a system or methodprovided herein ex vivo. In some embodiments, the treated cells areselected for those cells in which a desired nuclease-mediated genomicediting event has been effected. In some embodiments, treated cellscarrying a desired genomic mutation or alteration are returned to thesubject they were obtained from.

Methods for engineering, generation, and isolation of nucleasestargeting specific sequences, e.g., TALE, or zinc finger nucleases, andediting cellular genomes at specific target sequences, are well known inthe art (see, e.g., Mani et al., Biochemical and Biophysical ResearchCommunications 335:447-457, 2005; Perez et al., Nature Biotechnology26:808-16, 2008; Kim et al., Genome Research, 19:1279-88, 2009; Urnov etal., Nature 435:646-51, 2005; Carroll et al., Gene Therapy 15:1463-68,2005; Lombardo et al., Nature Biotechnology 25:1298-306, 2007;Kandavelou et al., Biochemical and Biophysical Research Communications388:56-61, 2009; and Hockemeyer et al., Nature Biotechnology27(9):851-59, 2009, as well as the reference recited in the respectivesection for each nuclease). The skilled artisan will be able toascertain suitable methods for use in the context of the presentdisclosure based on the guidance provided herein.

TALE Effector Proteins

In some embodiments, effector proteins comprising a TALE domain aredelivered to a target cell by a system or method provided herein. Insome embodiments, a TALE effector, e.g., an engineered TALEtranscription factor comprising a TALE DNA binding domain and aheterologous transcriptional activator or repressor domain, is deliveredto a cell by a system or method provided by aspects of this invention.In some embodiments, the TALE effector, e.g., a transcription factor, isdelivered to a cell in an amount sufficient to activate or inhibittranscription of a target gene of the transcription factor within thecell. In some embodiments, a transcription factor is delivered in anamount and over a time period sufficient to effect a change in thephenotype of a target cell, for example, a change in cellular function,or a change in developmental potential. Exemplary TALE transcriptionfactors are described herein, and the skilled artisan will be able toidentify additional suitable TALE transcription factors based on theguidance provided herein and the knowledge of such TALE transcriptionfactors in the art.

In some embodiments, a target cell, for example, a somatic cell, iscontacted with a TALE transcription factor, or a combination of suchfactors, associated with a supercharged protein provided herein. In someembodiments the target cell is a primary somatic cell and is contactedin vitro or ex vivo with a TALE transcription factor associated with asupercharged protein. In some embodiments, the TALE transcription factoris associated with a postiviely charged supercharged protein, e.g., asdescribed herein. In some embodiments, the TALE transcription factor isassociated with a negatively charged supercharged proteins, e.g., asdescribed herein. In some embodiments, the TALE transcription factor isassociated with a cationic lipid and/or cationic polymer, e.g., asdescribed herein. In some embodiments, the TALE transcription factor isassociated with a negatively charged supercharged protein and a cationiclipid and/or cationic polymer, e.g., as described herein.

In some embodiments, a target cell is contacted, or repeatedlycontacted, with a TALE transcription factor associated with asupercharged protein (and optionally a cationic lipid and/or cationicpolymer) as provided herein, and a desired change in cellular phenotypeor gene expression is detected. In some embodiments, a target cell iscontacted repeatedly with a TALE transcription factor associated with asupercharged protein (and optionally a cationic lipid and/or cationicpolymer) as provided herein until the formation of a desired cellularphenotype is detected. Methods for detecting cellular phenotypes andgene expression are well known to those in the art and include, forexample, morphological analysis, and detection of marker gene expressionby well-established methods such as immunohistochemistry, fluorescenceactivated cell sorting (FACS), or fluorescent microscopy. In someembodiments, a target cell is contacted with a TALE transcription factorassociated with a supercharged protein as provided herein for a periodof at least 3 hours, at least 6 hours, at least 12 hours, at least 1day, at least 2 days, at least 3 days, at least 4 days, at least 5 days,at least 6 days, at least 7 days, at least 10-12 days, at least 12-15days, at least 15-20 days, at least 20-25 days, at least 25-30 days, atleast 30-40 days, at least 40-50 days, at least 50-60 days, at least60-70, or at least 70-100 days.

In some embodiments, a target cell is contacted with a TALEtranscription factor associated with a supercharged protein (andoptionally a cationic lipid and/or cationic polymer) as provided hereinin an amount and for a time period effective to program the cell towardsa different cell state. As will be apparent to those of skill in theart, the amount necessary to program or re-program a cell will dependenton various factors, for example, on the cell type and the treatmentschedule. In general, delivery of a TALE transcription factor to atarget somatic cell by a system or method provided herein will be at aconcentration below a concentration at which significant toxicity can beobserved. The critical concentration will depend, for example, on thespecific TALE transcription factor, the supercharged protein it isassociated with, the type of association, and the type of cell beingtreated.

A useful concentration of a functional effector protein associated witha supercharged protein (and optionally a cationic lipid and/or cationicpolymer) for delivery to a specific cell type can be established bythose of skill in the art by routine experimentation. In someembodiments a target cell is contacted in vitro or ex vivo with afunctional effector protein associated with a supercharged protein (andoptionally a cationic lipid and/or cationic polymer) at a concentrationof about 1 pM to about 1 μM. In some embodiments, a target cell iscontacted in vitro or ex vivo with the functional effector proteinassociated to a supercharged protein at a concentration of about 1 pM,about 2.5 pM, about 5 pM, about 7.5 pM, about 10 pM, about 20 pM, about25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM,about 75 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about250 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about700 pM, about 750 pM, about 800 pM, about 900 pM, about 1 nM, about 2nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8nM, about 9 nM, about 10 nM, about 20 nM, about 25 nM, about 30 nM,about 40 nM, about 50 nM, about 60 nm, about 70 nM, about 75 nM, about80 nM, about 90 nM, about 100 nM, about 200 nM, about 250 nM, about 300nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 750nM, about 800 nM, about 900 nM, or about 1 μM. A useful time of exposureof the target cell to the functional effector protein, and, ifnecessary, incubation after administration in the absence of thefunctional effector protein, as well as a number ofadministration/incubation cycles useful to achieve a desired biologicaleffect (e.g., change in gene transcription, cleavage of a target site bya delivered nuclease, etc.), or a desired cellular phenotype can also beestablished by those of skill in the art by routine experimentation.

In some embodiments, the target cell for delivery of a functionaleffector protein by a system or method provided herein, is a primarycell obtained by a biopsy from a subject. In some embodiments, thesubject is diagnosed as having a disease. In some embodiments thedisease is a degenerative disease characterized by diminished functionof a specific cell type, for example, a neural cell. In someembodiments, a cell treated with a functional effector protein accordingto the strategies or methods disclosed herein, or the progeny of such acell, is used in a cell-replacement therapeutic approach. In someembodiments, the treated cells are administered to the subject fromwhich the somatic cell was obtained in an autologous cell replacementtherapeutic approach.

In some embodiments, a functional effector protein, e.g., TALEtranscription factor able to convert a cell from one differentiatedstate into another, is delivered to a target cell in vitro or in vivo bya system or method provided herein. Transcription factors that effecttransdifferentiation are known in the art (see, e.g., Zhou et al.,Nature 455:627-33, 2008). In some embodiments, a TALE transcriptionfactor modulating the expression of PPARγ or PRDM16 are delivered tofibroblast cells by a system or method as provided by this invention. Itis known in the art that expression these transcription factors is apivotal step in the programming of fibroblasts towards a brown fat orwhite fat cell state. In some embodiments, a programmed brown fat cellis generated from a fibroblast obtained from a subject in need of brownfat cells, and is administered to the subject, e.g., used in acell-replacement therapeutic approach involving the subject.

Formation of Complexes

The present invention provides complexes comprising superchargedproteins associated with one or more functional effector proteins to bedelivered. In some embodiments, supercharged proteins are associatedwith one or more functional effector proteins to be delivered throughnon-covalent interactions. In some embodiments, supercharged proteinsare associated with one or more functional effector proteins throughelectrostatic interactions. In certain embodiments, superchargedproteins have an overall net positive charge, and the functionaleffector proteins to be delivered have an overall net negative charge.In some embodiments, the complex further comprises a cationic lipidand/or cationic polymer. For example, in some embodiments, thesupercharged protein of the complex is supernegatively charged, allowingfor association with cationic lipids and/or polymers.

In certain embodiments, supercharged proteins are associated with one ormore functional effector proteins to be delivered via covalent bond. Forexample, a supercharged protein may be fused to a functional effectorprotein to be delivered. Covalent attachment may be direct or indirect(e.g., through a linker). In some embodiments, a covalent attachment ismediated through one or more linkers. In some embodiments, the linker isa cleavable linker. In certain embodiments, the cleavable linkercomprises an amide, ester, or disulfide bond. For example, the linkermay be an amino acid sequence that is cleavable by a cellular enzyme. Incertain embodiments, the enzyme is a protease. In other embodiments, theenzyme is an esterase. In some embodiments, the enzyme is one that ismore highly expressed in certain cell types than in other cell types.For example, the enzyme may be one that is more highly expressed intumor cells than in non-tumor cells. Exemplary linkers and enzymes thatcleave those linkers are presented below.

Cleavable Linkers

Linker Sequence Enzyme(s) Targeting Linker X¹-AGVF-X lysosomal thiolproteinases (see, e.g., Duncan et al., 1982, Biosci. (SEQ ID NO: 256)Rep., 2:1041-46; incorporated herein by reference) X-GFLG-X lysosomalcysteine proteinases (see, e.g., Vasey et al., Clin. Canc. (SEQ ID NO:257) Res., 1999, 5: 83-94; incorporated herein by reference) X-FK-XCathepsin B-ubiquitous, overexpressed in many solid tumors, such asbreast cancer (see, e.g., Dubowchik et al., 2002, Bioconjugate Chem.,13: 855-69; incorporated herein by reference) X-A*L-X CathepsinB-ubiquitous, overexpressed in many solid tumors, such as breast cancer(see, e.g., Trouet et al., 1982, Proc. Natl. Acad. Sci., USA, 79:626-29; incorporated herein by reference) X-A*LA*L-X CathepsinB-ubiquitous, overexpressed in many solid tumors (see, (SEQ ID NO: 258)e.g., Schmid et al., 2007, Bioconjugate Chem, 18: 702-16; incorporatedherein by reference) X-AL*AL*A-X Cathepsin D-ubiquitous (see, e.g.,Czerwinski et al., 1998, Proc. (SEQ ID NO: 259) Natl. Acad. Sci., USA,95: 11520-25; incorporated herein by reference) ¹X denotes asupercharged protein or a functional effector protein to be delivered*refers to observed cleavage site

To give but one particular example, a +36 GFP may be associated with afunctional effector protein to be delivered by a cleavable linker, suchas ALAL (SEQ ID NO:254), to generate +36GFP-(GGS)₄-ALAL-(GGS)₄-[functional effector protein X] (SEQ ID NO:255).

In certain embodiments, the functional effector protein to be deliveredis contacted with the supercharged protein to form a complex. In someembodiments, formation of complexes is carried out at or around pH 7. Insome embodiments, formation of complexes is carried out at about pH 5,about pH 6, about pH 7, about pH 8, or about pH 9. Formation ofcomplexes is typically carried out at a pH that does not negativelyaffect the function of the supercharged protein and/or the functionaleffector protein. In some embodiments, formation of complexes is carriedout at room temperature. In some embodiments, formation of complexes iscarried out at or around 37° C. In some embodiments, formation ofcomplexes is carried out below 4° C., at about 4° C., at about 10° C.,at about 15° C., at about 20° C., at about 25° C., at about 30° C., atabout 35° C., at about 37° C., at about 40° C., or higher than 40° C.Formation of complexes is typically carried out at a temperature thatdoes not negatively affect the function of the supercharged proteinand/or functional effector protein. In some embodiments, formation ofcomplexes is carried out in serum-free medium. In some embodiments,formation of complexes is carried out in the presence of CO₂ (e.g.,about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, or more).

In some embodiments, formation of complexes is carried out usingconcentrations of functional effector protein of about 100 nM. In someembodiments, formation of complexes is carried out using concentrationsof functional effector protein of about 25 nM, about 50 nM, about 75 nM,about 90 nM, about 100 nM, about 110 nM, about 125 nM, about 150 nM,about 175 nM, or about 200 nM. In some embodiments, formation ofcomplexes is carried out using concentrations of supercharged protein ofabout 40 nM. In some embodiments, formation of complexes is carried outusing concentrations of supercharged protein of about 10 nM, about 20nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM,about 80 nM, about 90 nM, or about 100 nM.

In some embodiments, formation of complexes is carried out underconditions of excess functional effector protein. In some embodiments,formation of complexes is carried out with ratios of functional effectorprotein:supercharged protein of about 20:1, about 10:1, about 9:1, about8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1,or about 1:1. In some embodiments, formation of complexes is carried outwith ratios of functional effector protein:supercharged protein of about3:1. In some embodiments, formation of complexes is carried out withratios of supercharged protein: functional effector protein of about20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1,about 4:1, about 3:1, about 2:1, or about 1:1.

In some embodiments, formation of complexes is carried out by mixingsupercharged protein with functional effector protein, and agitating themixture (e.g., by inversion). In some embodiments, formation ofcomplexes is carried out by mixing supercharged protein with functionaleffector protein, and allowing the mixture to sit still. In someembodiments, the formation of the complex is carried out in the presenceof a pharmaceutically acceptable carrier or excipient. In someembodiments, the complex is further combined with a pharmaceuticallyacceptable carrier or excipient. Exemplary excipients or carriersinclude water, solvents, lipids, proteins, peptides, endosomolyticagents (e.g., chloroquine, pyrene butyric acid), small molecules,carbohydrates, buffers, natural polymers, synthetic polymers (e.g.,PLGA, polyurethane, polyesters, polycaprolactone, polyphosphazenes),pharmaceutical agents, etc.

In some embodiments, complexes comprising supercharged protein andfunctional effector protein may migrate more slowly in gelelectrophoresis assays than either the supercharged protein alone or thefunctional effector protein alone.

Applications

The present invention provides compositions comprising superchargedproteins, naturally occurring or engineered, associated with functionaleffector proteins (e.g., nucleases, transcriptionalactivators/repressors, recombinases, Cas9 proteins including variantsand fusions thereof, etc.) to be delivered to a cell, as well as methodsof using such compositions and uses of such compositions. In certainembodiments, compositions are provided comprising a Cas9 protein (e.g.,wherein the Cas9 protein is associated with a gRNA) and a cationiclipid. In certain embodiments, compositions are provided comprising aCas9 protein (e.g., wherein the Cas9 protein is associated with a gRNA)and a cationic polymer. The inventive compositions may be used to treator prevent any disease that can benefit, e.g., from the delivery of anagent to a cell. The inventive compositions may also be used totransfect or treat cells for research purposes.

In some embodiments, compositions in accordance with the invention maybe used for research purposes, e.g., to efficiently deliver functionaleffector proteins to cells in a research context. In some embodiments,compositions in accordance with the present invention may be used fortherapeutic purposes. In some embodiments, compositions in accordancewith the present invention may be used for treatment of any of a varietyof diseases, disorders, and/or conditions, including, but not limitedto, one or more of the following: autoimmune disorders (e.g., diabetes,lupus, multiple sclerosis, psoriasis, rheumatoid arthritis);inflammatory disorders (e.g., arthritis, pelvic inflammatory disease);infectious diseases (e.g., viral infections (e.g., HIV, HCV, RSV),bacterial infections, fungal infections, sepsis); neurological disorders(e.g. Alzheimer's disease, Huntington's disease; autism; Duchennemuscular dystrophy); cardiovascular disorders (e.g. atherosclerosis,hypercholesterolemia, thrombosis, clotting disorders, angiogenicdisorders such as macular degeneration); proliferative disorders (e.g.cancer, benign neoplasms); respiratory disorders (e.g. chronicobstructive pulmonary disease); digestive disorders (e.g. inflammatorybowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia,arthritis); endocrine, metabolic, and nutritional disorders (e.g.diabetes, osteoporosis); urological disorders (e.g. renal disease);psychological disorders (e.g. depression, schizophrenia); skin disorders(e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia,hemophilia); etc.

Compositions of the invention may be used in a clinical setting. Forexample, a supercharged protein may be associated with a functionaleffector protein that can be used for therapeutic applications. Suchfunctional effector protein may be, for example, nucleases ortranscriptional activators. Other compositions comprising a Cas9 proteinand a cationic lipid may also be used for therapeutic applications.

In some embodiments, the supercharged protein or functional effectorprotein associated with a supercharged protein includes a detectablelabel. These molecules can be used in detection, imaging, diseasestaging, diagnosis, or patient selection. Suitable labels includefluorescent, chemiluminescent, enzymatic labels, colorimetric,phosphorescent, density-based labels, e.g., labels based on electrondensity, and in general contrast agents, and/or radioactive labels.

Pharmaceutical Compositions

The present invention provides compositions comprising superchargedproteins associated with at least one functional effector protein to bedelivered, and in some embodiments are encapsulated by cationic lipids.Other compositions comprising a Cas9 protein and a cationic lipid areprovided. Thus, the present invention provides pharmaceuticalcompositions comprising one or more supercharged proteins associatedwith a functional effector protein, and/or one or more functionaleffector proteins associated with a cationic lipid and/or cationicpolymer, and one or more pharmaceutically acceptable excipients.Pharmaceutical compositions may optionally comprise one or moreadditional therapeutically active substances. In accordance with someembodiments, a method of administering pharmaceutical compositionscomprising one or more supercharged proteins associated with afunctional effector protein to be delivered to a subject in need thereofis provided. In some embodiments, compositions are administered tohumans. For the purposes of the present disclosure, the phrase “activeingredient” generally refers to a Cas9 protein and/or superchargedprotein associated with a functional effector protein, or to thefunctional effector protein to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, including commercially relevant mammalssuch as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;and/or birds, including commercially relevant birds such as chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate (Tween®20), polyoxyethylene sorbitan (Tween®60),polyoxyethylene sorbitan monooleate [Tween®80], sorbitan monopalmitate[Span®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [Span®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether (Brij®30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GlydantPlus®, Phenonip®, methylparaben, Germall®115, Germaben®II, Neolone™,Kathon™, and/or Euxyl®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices. Intradermalcompositions may be administered by devices which limit the effectivepenetration length of a needle into the skin and functional equivalentsthereof. Jet injection devices which deliver liquid compositions to thedermis via a liquid jet injector and/or via a needle which pierces thestratum corneum and produces a jet which reaches the dermis aresuitable. Ballistic powder/particle delivery devices which usecompressed gas to accelerate vaccine in powder form through the outerlayers of the skin to the dermis are suitable. Alternatively oradditionally, conventional syringes may be used in the classical mantouxmethod of intradermal administration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder and/or using a self-propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Administration

The present invention provides methods comprising administeringcompositions of supercharged proteins associated with functionaleffector proteins to a subject in need thereof. In some embodiments,methods of administering compositions comprising other functionaleffector proteins (e.g., a Cas9 protein) and cationic lipid and/orcationic polymers are provided. Such compositions may be administered toa subject using any amount and any route of administration effective forpreventing, treating, diagnosing, or imaging a disease, disorder, and/orcondition. The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

Compositions of supercharged proteins associated with functionaleffector proteins to be delivered as well as compositions comprisinge.g., a Cas9 protein and cationic lipid may be administered by anyroute. In some embodiments, such compositions are administered by one ormore of a variety of routes, including oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, subcutaneous,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical (e.g., by powders, ointments, creams, gels,lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal,intratumoral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; as an oral spray, nasal spray, and/oraerosol, and/or through a portal vein catheter. In some embodiments,supercharged proteins or complexes, and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof, are administered bysystemic intravenous injection. In specific embodiments, superchargedproteins or complexes and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof may be administered intravenously and/ororally. In specific embodiments, such compositions may be administeredin a way which allows the functional effector protein to cross theblood-brain barrier, vascular barrier, or other epithelial barrier.

In certain embodiments, compositions in accordance with the inventionmay be administered at dosage levels sufficient to deliver an amount offunctional effector protein of from about 0.0001 mg/kg to about 100mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

Compositions comprising supercharged proteins associated with functionaleffector proteins may be administered in combination with one or moreother therapeutic, prophylactic, diagnostic, or imaging agents. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope of theinvention. Compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, theinvention encompasses the delivery of pharmaceutical, prophylactic,diagnostic, or imaging compositions in combination with agents that mayimprove their bioavailability, reduce and/or modify their metabolism,inhibit their excretion, and/or modify their distribution within thebody.

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments. In some embodiments, kits comprise one ormore of (i) a supercharged protein, as described herein; (ii) afunctional effector protein to be delivered; (ii) a cationic lipidand/or cationic polymer; and (iv) instructions for formulating acomposition comprising the functional protein associated to thesupercharged protein. In some embodiments, the kits comprise a Cas9protein and a cationic lipid. In some embodiments, kits comprise anucleic acid encoding for the supercharged protein and/or the functionalprotein to be delivered. In some embodiments, the kit comprises acloning vector encoding a supercharged protein and a cloning siteallowing the in-frame cloning of a functional effector protein togenerate a fusion protein. In some embodiments, kits comprise apharmaceutical composition provided herein comprising a superchargedprotein associated with a functional effector protein; a syringe,needle, or applicator for administration of the pharmaceuticalcomposition to a subject; and instructions for administration of thepharmaceutical composition to the subject.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1: Delivery of TALE Activators Fused to SuperchargedGFP

A major target for reprogramming fibroblast cell fate towards brown orwhite adipocyte cell fate lies in the switch from White Adipose Tissue(WAT) to Brown Adipose Tissue (BAT), which is governed by expression ofPRDM16 and PPARγ. Robust TALE transcriptional activators fused to a +36GFP were engineered that target PPARγ and PRDM16 genomic sequences infibroblasts. Fusion proteins were purified using a heparin column and/oran SEC and gels show a single band at 130 kD The modulation ofexpression and effect on cellular phenotype after delivery of the TALEactivators was compared to the modulation after viral delivery of aPPARγ cDNA followed by 7-day treatment with adipogenesis cocktail. Itwas observed that adipocytes formed upon treatment with +36 GFPTALEPRDM16 fusion. Expression of white adipose tissue marker genes wasdetected after delivery of supercharged PRDM16 TALE activators.

A one-time supercharged protein-mediated delivery of a TALE activatorfor PPARγ was found to induce expression of white-fat genes and todifferentiate fibroblasts into white-fat cells. Superchargedprotein-mediated delivery of both a PPARγ and PRDM16 TALE activatorinduced the differentiation of fat cells with increased expression ofbrown-fat markers such as PRDM16, cox8b, elovl3, and cidea as well as asmall increase in thermogenic gene expression markers PGC1a and UCP1.

An Aurein peptide was fused to the N-terminus of the+36GFP-TALE-activator fusion protein. Delivery of Aurein +36 GFP TALEpurified by heparin column was observed by detecting fluorescence innucleus of the treated cells.

FIG. 1 shows a schematic of macromolecular delivery into mammaliancells. FIG. 2 shows an overview of the switch from White Adipose Tissue(WAT) to Brown Adipose Tissue (BAT). FIG. 3 shows a schematic ofsupercharged delivery platforms to deliver TALE activators programmed totarget PPARγ or PRDM16. FIG. 4 shows a schematic of a fusion proteincomprising a +36 GFP fusion, an 18.5 mer TALE domain, and a VP64activation domain. FIG. 5 shows expression and purification of the +36GFP-TALE activator-fusion protein. FIG. 6 shows testing assays foractivation of fat cell regulator genes upon delivery of +36 GFP PPARγand PRDM16 TALE activator fusion proteins.

FIG. 7 shows delivery efficacy of +36 GFP TALE activator fusion proteinsat different concentrations. FIG. 8 shows a comparison of deliveryefficacy of two different +36 GFP-PRDM16 TALE fusion proteins in NIH 3T3cells. FIG. 9 shows PPARγ gene expression after delivery of PPARγ-TALEactivator fusion and comparison to various controls. FIG. 10 showsPRDM16 gene expression after delivery of RDM16-TALE activator fusion andcomparison to various controls. FIG. 11 shows moderate TALE activityobserved in the presence of serum.

FIG. 12 shows a validation of viral delivery of PPARγ followed by 7-daytreatment with adipogenesis cocktail. FIG. 13 shows a schematic of anassay for programming fibroblasts into WAT and BAT. FIG. 14 showsadipocyte formation observed upon treatment with +36 GFP TALE activatorfusion protein. FIG. 15 shows staining of various treatments after 7days with LipidTOX red, demonstrating formation of adipocytes afterviral delivery as well as after delivery of supercharged PPARγ TALEactivator fusion protein. FIG. 16 shows staining of cells after varioustreatments after 7 days with LipidTOX red, demonstrating formation ofadipocytes after viral delivery as well as after delivery ofsupercharged PPARγ TALE activator fusion protein. FIG. 17 showsexpression of WAT biomarker genes after viral delivery as well as afterdelivery of supercharged PPARγ TALE activator fusion protein.

Example 2: In Vivo Delivery of TALE Activators Fused to Supercharged GFP

NIH 3T3 cells were grown to 70-90% confluence and treated with 1 μM orbetween 0.5-5 μM of +36 GFP PPARγ TALE and/or +36 GFP PRDM16 TALE fusionprotein in DMEM without serum. A serum-free medium was chosen, becauseserum can decrease the effectiveness of protein-based delivery. Cellswere incubated with the respective fusion protein solution for 4 hoursbefore the media was removed and full DMEM containing serum was addedback to cells. Control cells were infected with a viral constructencoding PPARγ or PRDM16 in order to serve as a positive control forexpression of WAT and BAT genes according to known protocols (see, e.g.,Seale et al. 2008 Nature 454, 961-967, the entire contents of which areincorporated herein by reference). Once all cells reached 100%confluence an adipogenesis cocktail containing isobutylmethylxanthine,insulin, rosiglitazone, dexamethasone, T3, and indomethacin was added tothe cells and replaced 48 hours later with a form of the cocktailcontaining only insulin, T3, and rosiglitazone. At 48 hours after thissecond replacement of cocktail another dosage of T3, insulin, androsiglitazone was added to the cells. The next day, which is now oneweek from the start of the experiment, cells were harvested with TRIzol,total RNA was extracted, and qRT-PCR was performed to measure geneexpression levels of PPARγ, PRDM16, and other brown fat marker genessuch as UCP1, PGC1a, Elovl3, and Cidea.

FIG. 18 shows delivery of supercharged PRDM16 TALE activator fusionproteins to induce brown-fat adipocytes in vivo. Robust adipocyteformation was observed after viral delivery of PPARγ and PRDM16 and alsoafter delivery of supercharged TALE activator protein fusions. FIG. 19shows a comparison of supercharged (TALE) and viral delivery of PPARγand PRDM16 to cells. The figure shows TALE/TALE, viral/TALE, andviral/viral-induced expression of brown fat markers by expression ofPPARγ and PRDM16. FIG. 20 shows RT-qPCR assessments consistent with fatcell differentiation, which were also observed by LipidTOX staining.

Example 3: Delivery of TALE Activators Complexed with Supercharged GFP

In order to improve delivery efficacy, protein complexes in which thefunctional protein was non-covalently associated with the superchargedprotein were generated and administered to cells. FIG. 21. shows thatdelivery of functional TALE activator fusion proteins as complexes with+36 GFP improves TALE activator activity after delivery. FIG. 22 showsPRDM16 gene expression after TALE activator fusion delivery either as afusion (+36GFP PRDM16 TALE-3) or a complex (+36GFP+PRDM16 TALE-3) with+36GFP. It was observed that delivery of complexes tended to increaseTALE activator activity.

Example 4: Effect of Aurein Fusions on Delivery Efficacy

FIG. 23 shows the effect of an N-terminal Aurein peptide fusion to+36GFP on PRDM16 gene expression after TALE activator fusion delivery(either as a fusion or a complex with +36GFP). The Aurein peptide wasfused to the N-terminus of the GFP-TALE construct via a GGS(9) (SEQ IDNO:252) linker, resulting in an Aurein peptide-GGS(9) linker-(+)36GFP-protein-GGS(9) linker −PRDM16 TALE-3 fusion protein. The protein waspurified using size exclusion chromatography.

Example 5: Delivery of TALE Activators Complexed with Supercharged GFPor Cationic Lipids

FIG. 24 shows PRDM16 gene expression after TALE PRDM16 activator proteindelivery either as a fusion with +36 GFP (+36GFP PRDM16 TALE-3), acomplex with +36 GFP (+36GFP+PRDM16 TALE-3), or a complex withLipofectamine LTX, for which an increase in gene expression wasobserved.

Example 6: Delivery of Cas9 Fused to Supercharged GFP

Supercharged delivery of Cas9 into mammalian cells would allow theapplication of powerful RNA-programmable nuclease technology in cellswithout the drawbacks of prior delivery methods. To this end, a Cas9fusion with +36GFP was generated, using an ALAL linker. FIG. 25 shows aschematic of the supercharged fusion protein with Cas9. FIG. 26 showsthe purification of wild-type Cas9 protein and Cas9 fusion proteins with+36GFP and Aurein-GGS9. The fusion protein is administered to cells inthe same manner as the TALE activator fusion proteins above. The Cas9,once delivered to the cells, binds and cleaves its target site in thecellular genome. Nuclease activity in the target cells is detected via asuitable assay, e.g., via southern blot or sequencing assay.

Example 7: Efficient Delivery of Genome Editing Proteins In Vitro and InVivo

Efficient intracellular delivery of proteins to the nucleus or cytoplasmis needed to fully realize the potential of protein therapeuticsincluding genome-editing agents. Current methods of protein deliveryoften suffer from low tolerance for serum proteins, poor endosomalescape, and limited in vivo efficacy. As demonstrated in this Example,common cationic lipid reagents originally developed for nucleic acidtransfection can potently deliver proteins that are fused to negativelysupercharged proteins, that contain natural anionic domains, or thatnatively bind to anionic nucleic acids. This approach mediates thefunctional delivery of Cre recombinase, TALE- and Cas9-basedtranscriptional activators, and Cas9:sgRNA nuclease complexes intocultured human cells at low nanomolar concentrations in media containing10% serum. Lipid-based delivery can be >1,000-fold more potent thancationic protein delivery strategies. Delivery of Cas9:sgRNA complexesresulted in genome modification efficiencies as high as 80% withsubstantially higher specificity compared to standard DNA transfection,likely due to the transient nature of delivered Cas9:sgRNA complexes.This approach also mediated efficient delivery of Cre recombinase andCas9:sgRNA complexes into the mouse inner ear in vivo, achieving up to90% Cre-mediated recombination and 20% Cas9-mediated genome modificationin the targeted hair-cell population.

Materials and Methods

Construction of Cas9, Cre, and TALE Fusion and sgRNA ExpressionPlasmids.

Sequences of all constructs used in this paper are listed below orprovided elsewhere in the specification. All protein constructs weregenerated from previously reported plasmids for protein of interestcloned into a pET29a expression plasmid.

Expression and Purification of S. pyogenes Cas9 and Other Proteins.

E. coli BL21 STAR (DE3) competent cells (Life Technologies) weretransformed with pMJ806⁴⁷ encoding the S. pyogenes Cas9 fused to anN-terminal 10×His-tag/maltose binding protein. The resulting expressionstrain was inoculated in Luria-Bertani (LB) broth containing 100 μg/mLof ampicillin at 37° C. overnight. The cells were diluted 1:100 into thesame growth medium and grown at 37° C. to OD₆₀₀=˜0.6. The culture wasincubated at 20° C. for 30 min, and isopropylβ-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce Cas9expression. After approximately 16 hours, the cells were collected bycentrifugation at 8,000 g and resuspended in lysis buffer (50 mMtris(hydroxymethyl)-aminomethane (Tris)-HCl, pH 8.0, 1 M NaCl, 20%glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP)). The cells werelysed by sonication (1 sec pulse-on, 1 sec pulse-off for 15 minutestotal at 6 W output) and the soluble lysate was obtained bycentrifugation at 20,000 g for 30 minutes.

The cell lysate was incubated with His-Pur nickel-nitriloacetic acid(nickel-NTA) resin (Thermo Scientific) at 4° C. for 30 minutes tocapture His-tagged Cas9. The resin was transferred to a 20-mL column andwashed with 20 column volumes of lysis buffer. Cas9 was eluted in 50 mMTris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, 10 mM TCEP, and 300 mMimidazole, and concentrated by Amicon ultra centrifugal filter(Millipore, 100-kDa molecular weight cut-off) to ˜50 mg/mL. The 6×Histag and maltose-binding protein were removed by TEV protease treatmentat 4° C. for 20 hours and captured by a second Ni-affinity purificationstep. The eluent, containing Cas9, was injected into a HiTrap SP HPcolumn (GE Healthcare) in purification buffer containing 50 mM Tris-HCl(pH 8), 0.1 M NaCl, 20% glycerol, and 10 mM TCEP. Cas9 was eluted withpurification buffer containing a linear NaCl gradient from 0.1 M to 1 Mover five column volumes. The eluted fractions containing Cas9 wereconcentrated down to a concentration of 200 μM as quantified byBicinchoninic acid assay (BCA) (Pierce Biotechnology), snap-frozen inliquid nitrogen, and stored in aliquots at −80° C. All other proteinswere purified by this method but without TEV cleavage step and proteinscontaining (−30)GFP were purified by anion exchange using a Hi-Trap Q HPanion exchange column (GE Healthcare) using the same purificationprotocol.

In Vitro Transcription of sgRNAs.

Linear DNA fragments containing the T7 promoter binding site followed bythe 20-bp sgRNA target sequence were transcribed in vitro using the T7High Yield RNA Synthesis Kit (NEB) according to the manufacturer'sinstructions. In vitro transcribed RNA was precipitated with ethanol andpurified by gel electrophoresis on a Criterion 10% polyacrylamideTBE-Urea gel (Bio-Rad). Excised gel fragments were extracted in 420 μLof 300 mM NaCl overnight on a rocking surface at 4° C. Gel-purifiedsgRNA was precipitated with ethanol and redissolved in water and sgRNAconcentration was finally quantified by UV absorbance and snap-frozen at−80° C.

Plasmid Transfection.

Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies)according the manufacturer's protocol. For TALE activator plasmids, 300ng of DNA was transfected, and for the activator synergy experiments 60ng of each of five plasmids was pooled and transfected. For Cas9nuclease delivery experiments, linear DNA PCR products expressing sgRNAswere used in transfection experiments targeting genomic sites in CLTA,EMX, VEGF, and GFP (sgRNA GFP g1, GFP g3, GFP g5, and GFP g7 for nickasestudies). Linear DNA PCR products were generated using plasmidcontaining the U6 promoter as template and forward primers bearing theU6 promoter upstream sequence and reverse primers containing U6downstream sequence followed by the sgRNA sequence (20-bp sequenceunique to each target plus constant sgRNA backbone architecturesequence). sgRNAs expressed from linear DNA templates contained at leasttwo 5′ guanosines to match in vitro transcribed sgRNAs that requiredthese bases for T7 transcription. Primer sequences and PCR conditionsare listed below. For dCas9 activator experiments, 700 ng of Cas9 ordCas9-VP64 plasmid DNA was co-transfected with 250 ng of the appropriatesgRNA expression plasmid. For activator synergy experiments 50 ng of DNAfrom each of the six sgRNA was pooled and co-transfected with 700 ng ofdCas9-VP64 plasmid.

Delivery of Transcription Factor Proteins Complexed with Cationic Lipidsin Cell Culture.

A more in-depth description of the delivery of genome-editing proteinsboth in vitro and in vivo can be found below. Briefly, cultured cellswere plated in 48-well format (250 μL volume) in Dulbecco's ModifiedEagle's Media plus GlutaMAX (Life Technologies, Carlsbad, Calif.) with10% FBS (“full serum media”) and antibiotics at a cell density necessaryto reach ˜70% confluence the next day. Full serum media was replacedwith the same media but containing no antibiotics one hour beforedelivery. Delivery of Cre and TALE proteins was performed by combining 1nM to 1 μM protein (in 275 μL final volume) with 0.5-1.5 μL ofcommercially available cationic lipids in 25 μL OPTIMEM media (LifeTechnologies, Carlsbad, Calif.) according to the manufacturer's protocolfor normal plasmid transfection, including incubation time. For Cas9delivery in vitro, transcribed sgRNA was incubated with Cas9 protein for5 min before complexing with the cationic lipid reagent. 25 μL lipidcomplexes in OPTIMEM media were added to cells and media was replaced12-16 hours later fresh media unless otherwise noted. Cells were assayedfor recombination 48 hours after delivery, for gene activation either 4or 16 hours after delivery, and for gene modification 72 hours afterdelivery.

T7 Endonuclease I Assay to Detect Genomic Modifications.

U2OS-EGFP cells or HEK293T cells were transfected with Cas9 expressionand sgRNA expression plasmids or linear DNA PCR products as describedabove or treated with only Cas9 protein, only in vitro transcribedsgRNA, or only RNAiMAX. Genomic DNA was isolated from cells 2 days aftertransfection using the DNAdvance Kit (Agencourt) following themanufacturer's instructions. 200 ng of genomic DNA was used as templatein PCR reactions to amplify the targeted genomic loci with flankingsurvey primer pairs specified below. PCR products were purified with aQIAquick PCR Purification Kit (Qiagen) and quantified with Quant-iT™PicoGreen® dsDNA Kit (Life Technologies). 250 ng of purified PCR DNA wascombined with 2 μL of NEBuffer 2 (NEB) in a total volume of 19 μL anddenatured then re-annealed with thermocycling at 95° C. for 5 minutes,95 to 85° C. at 2° C./s; 85 to 20° C. at 0.2° C./s. The re-annealed DNAwas incubated with 1 μl of T7 Endonuclease I (10 U/μl, NEB) at 37° C.for 15 minutes. 10 μL of 50% glycerol was added to the T7 Endonucleasereaction and 12 μL was analyzed on a 5% TBE 18-well Criterion PAGE gel(Bio-Rad) electrophoresed for 30 minutes at 200 V, then stained with1×SYBR Gold (Life Technologies) for 30 min. Cas9-induced cleavage bandsand the uncleaved band were visualized on an AlphaImager HP (AlphaInnotech) and quantified using ImageJ software⁵⁴. The peak intensitiesof the cleaved bands were divided by the total intensity of all bands(uncleaved+cleaved bands) to determine the fraction cleaved which wasused to estimate gene modification levels as previously described.⁴⁶ Foreach sample, transfections and subsequent modification measurements wereperformed in triplicate on different days.

Stem Cell Culture and Delivery.

Mouse embryonic stem cell (ES) line Tau-GFP containing a permanent GFPgene insertion was cultured in DMEM with 15% FBS (Gibco), 100 mM MEMnonessential amino acids (Gibco), 0.55 mM 2-mercaptoethanol, andleukemia inhibitory factor (1,000 units/ml; Chemicon). After 5 daysfloating spheres were formed that exhibited GFP fluorescence. Complexesof Cas9:sgRNA and RNAiMAX were added to the culture containing thefloating spheres for 16 hours. After Cas9:sgRNA treatment, the cellswere cultured in the above media for 3 days. The floating spheres weretreated with trypsin for 5 minutes then passed through a 70 μm filter tocollect single cells. The cells were cultured on laminin-coated slidesin DMEM/F12 (1:1) supplemented with 1×N2, 1×B27, penicillin-streptomycin(100 μg/mL) and 10% FBS for two days before labeling.Immunohistochemistry was performed using an anti-GFP antibody (#ab13970,Abcam) to assess GFP expression. To quantify the number of GFP-negativecells, we counted the total number of GFP-positive and GFP-negativecells from three representative visual fields at 20× magnification, andcalculated the average efficiency. Three independent experiments wereperformed for each condition.

Microinjection of Proteins to Mouse Inner Ear.

P0 floxP-tdTomato mice were used for (−30)GFP-Cre injection and P2Atoh1-GFP mice were used for Cas9:sgRNA injection. Animals were usedunder protocols approved by the Massachusetts Eye & Ear Infirmary ALCUCcommittee. Mice were anesthetized by lowering their temperature on ice.Cochleostomies were performed by making an incision behind the ear toexpose the otic bulla. Glass micropipettes held by a micromanipulatorwere used to deliver the complex into the scala media, which allowsaccess to inner ear hair cells. For delivery of (−30)GFP-Cre, 3 μL of 45μM protein was mixed with 3 μL of either RNAiMAX or Lipofectamine 2000and incubated at room temperature for 30 minutes prior to injection.Four mice were injected per treatment group. For delivery of Cas9:sgRNAcomplexes, 1 μL of 200 μM Cas9 protein was mixed with 2 μL of 100 μMsgRNA and incubated for 5 minutes at room temperature before mixing with3 μL of either RNAiMAX or Lipofectamine 2000 and incubating for anadditional 30 minutes prior to injection. Three mice were injected pertreatment group. The total delivery volume for every injection was 0.3μL per cochlea and the release was controlled by a micromanipulator atthe speed of 32 mL/sec.

Immunohistochemistry and Quantification.

5-10 days after injection, the mice were sacrificed and cochlea wereharvested by standard protocols.⁵⁵ For immunohistochemistry, antibodiesagainst hair-cell markers (Myo7a and Esp) and supporting cells (Sox2)were used following a previously described protocol.⁵⁵ To quantify thenumber of tdTomato positive cells after (−30)GFP-Cre or GFP negativecells after Cas9:sgRNA delivery, we counted the total number of outerhair cells in a region spanning 200 μm around the site of injection inthe base turn of the cochlea. The efficiency of (−30)GFP-Cre-inducedrecombination or Cas9:sgRNA-induced genome modification was calculatedas the percentage of outer hair cells that expressed tdTomato or thatlost GFP expression.

High-Throughput DNA Sequencing of Genome Modifications.

HEK293T cells were either transfected with Cas9 and sgRNA expressionplasmids or linear DNA PCR products or treated with 50 nM Cas9 protein,250 nM purified sgRNA, and cationic lipids as described earlier for Cas9protein delivery to U2OS-EGFP reporter cells. For plasmid-basedtransfection experiments, 700 ng of Cas9 expression plasmid plus 250 ngof sgRNA plasmid or 50 ng of a linear DNA PCR product expressing sgRNAfor targeting either the EMX1, CLTA2, or VEGF locus were transfectedwith Lipofectamine 2000 (Life Technologies) and cells were isolated 2days later. For protein delivery experiments in vivo, ˜30 mg of mousetissue was isolated as previously described⁵⁵ from anesthetized mice andgenomic DNA was extracted using the Agencourt DNAAdvance Genomic DNAIsolation Kit (Beckman Coulter). For cell culture experiments genomicDNA was isolated as described above. 150 ng of genomic DNA was used astemplate to amplify by PCR the on-target and off-target genomic siteswith flanking HTS primer pairs specified below. Relative amounts ofcrude PCR products were quantified by gel electrophoresis and samplestreated with different sgRNA pairs or Cas9 nuclease types wereseparately pooled in equimolar concentrations before purification withthe QIAquick PCR Purification Kit (Qiagen). Approximately 150 ng ofpooled DNA was electrophoresed using a 5% TBE 18-well Criterion PAGE gel(BioRad) for 30 min at 200 V and DNAs ˜125 bp to ˜300 bp in length wereisolated and purified by QIAquick PCR Purification Kit (Qiagen).Purified DNA was amplified by PCR with primers containing sequencingadapters, purified, and sequenced on a MiSeq high-throughput DNAsequencer (Illumina) as previously described.⁴⁷

Data Analysis

Illumina sequencing reads were filtered and parsed with scripts writtenin Unix Bash as outlined below. Sample sizes for sequencing experimentswere maximized (within practical experimental considerations) to ensuregreatest power to detect effects. Statistical analyses for Cas9-modifiedgenomic sites (Table 2) were performed as previously described⁵⁶ withmultiple comparison correction using the Bonferroni method.

The following is a list of upstream and downstream flanking sequencesfor each genomic target site.

Target Downstream genomic Upstream genomic Site sequence sequence EMX_OnGGCCTGCTTCGTGGCAATGC ACCTGGGCCAGGGAGGGAGG (SEQ ID NO: 119)(SEQ ID NO: 120) EMX_Off1 CTCACTTAGACTTTCTCTCC CTCGGAGTCTAGCTCCTGCA(SEQ ID NO: 121) (SEQ ID NO: 122) EMX_Off2 TGGCCCCAGTCTCTCTTCTACAGCCTCTGAACAGCTCCCG (SEQ ID NO: 123) (SEQ ID NO: 124) EMX_Off3TGACTTGGCCTTTGTAGGAA GAGGCTACTGAAACATAAGT (SEQ ID NO: 125)(SEQ ID NO: 126) EMX_Off4 TGCTACCTGTACATCTGCAC CATCAATGATTGGGCATTTC(SEQ ID NO: 127) (SEQ ID NO: 128) VEG_On ACTCCAGTCCCAAATATGTAACTAGGGGGCGCTCGGCCAC (SEQ ID NO: 129) (SEQ ID NO: 130) VEG_Off1CTGAGTCAACTGTAAGCATT GGCCAGGTGCAGTGATTCAT (SEQ ID NO: 131)(SEQ ID NO: 132) VEG_Off2 TCGTGTCATCTTGTTTGTGC GGCAGAGCCCAGCGGACACT(SEQ ID NO: 133) (SEQ ID NO: 134) VEG_Off3 CAAGGTGAGCCTGGGTCTGTATCACTGCCCAAGAAGTGCA (SEQ ID NO: 135) (SEQ ID NO: 136) VEG_Off4TTGTAGGATGTTTAGCAGCA ACTTGCTCTCTTTAGAGAAC (SEQ ID NO: 137)(SEQ ID NO: 138) CLT2_On CTCAAGCAGGCCCCGCTGGT TTTTGGACCAAACCTTTTTG(SEQ ID NO: 139) (SEQ ID NO: 140) CLT2_Off1 TGAGGTTATTTGTCCATTGTTAAGGGGAGTATTTACACCA (SEQ ID NO: 141) (SEQ ID NO: 142) CLT2_Off2TCAAGAGCAGAAAATGTGAC CTTGCAGGGACCTTCTGATT (SEQ ID NO: 143)(SEQ ID NO: 144) CLT2_Off3 TGTGTGTAGGACTAAACTCT GATAGCAGTATGACCTTGGG(SEQ ID NO: 145) (SEQ ID NO: 146) EGFP AGCGTGTCCGGCGAGGGCGAAGCGTGTCCGGCGAGGGCGA (SEQ ID NO: 147) (SEQ ID NO: 148) MusEMXCAGAATCGGAGGACAAAATA ACGAAGCAGGCCAACGGGGA CAAAC GGACA (SEQ ID NO: 149)(SEQ ID NO: 150)Primers Used for Generating PCR Products to Serve as Substrates for T7Transcription of sgRNAs.

T7_gRNA-Rev was used in all cases. DNA template used was EGFP sgRNAplasmid as noted above. NTF3 and VEGF sgRNAs for dCas9-VP64 activatorexperiments were reported previously (Maeder et al., CRISPR RNA-guidedactivation of endogenous human genes. Nat. Methods. 2013; 10, 977-979).

(SEQ ID NO: 151) T7_EGFP1-Fwd TAA TAC GAC TCA CTA TA GGGCACGGGCAGCTTGCCGG (SEQ ID NO: 152)T7-GFP g1-Fwd TAA TAC GAC TCA CTA TA GGCCTCGAACTTCACCTCGGCGGAAAGGACGAAACACC (SEQ ID NO: 153)T7-GFP g5-Fwd TAA TAC GAC TCA CTA TA GGCTGAAGGGCATCGACTTCAGAAAGGACGAAAC ACC (SEQ ID NO: 154)T7-GFP g3-Fwd TAA TAC GAC TCA CTA TA GGCAGCTCGATGCGGTTCACCAGAAAGGACGAAACACC (SEQ ID NO: 155)T7-GFP g7-Fwd TAA TAC GAC TCA CTA TA GGCAAGGAGGACGGCAACATCCGAAAGGACGAAACACC (SEQ ID NO: 156)T7-EMX-Fwd TAA TAC GAC TCA CTA TA GGAGTCCGAGCAGAAG AAGAAGAAAGGACGAAACACC(SEQ ID NO: 157) T7-VEG-Fwd TAA TAC GAC TCA CTA TA GGGGTGGGGGGAGTTTGCTCCGAAAGGACGAAACACC (SEQ ID NO: 158)T7-CLT2-Fwd TAA TAC GAC TCA CTA TA GGCAGATGTAGTGTTTCCACAGAAAGGACGAAACACC (SEQ ID NO: 159)T7_gRNA-Rev AAAAAAAGCACCGACTCGGTGPrimers for Generating Linear DNA PCR Product for Transfection.

PCR extension at (72° C., 3 min) on plasmid containing U6 promoter astemplate with PCR_sgRNA-fwd1, PCR_sgRNA-rev2 and appropriate PCR_sgRNAprimers listed below.

PCR_gRNA-fwd1 (SEQ ID NO: 160) CTGTACAAAAAAGCAGGCTTTA PCR_gRNA-rev2(SEQ ID NO: 161) AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC PCR-G-GFP1 (SEQ ID NO: 162)GAAAGGACGAAACACC GGCCTCGAACTTCACCTCGGCGGTTTTAGAGCTAGAAATAGCAA PCR-G-GFP3(SEQ ID NO: 163) GAAAGGACGAAACACCGGCAGCTCGATGCGGTTCACCAGTTTTAGAGCTAGAAATAGCAA PCR-G-GFP5 (SEQ ID NO: 164)GAAAGGACGAAACACC GGCTGAAGGGCATCGACTTCAGTTTTAGAGCTAGAAATAGCAA PCR-G-GFP7(SEQ ID NO: 165) GAAAGGACGAAACACCGGCAAGGAGGACGGCAACATCCGTTTTAGAGCTAGAAATAGCAA PCR-G-CLT2 (SEQ ID NO: 166)GAAAGGACGAAACACC GGCAGATGTAGTGTTTCCACAGTTTTAGAGCTAGAAATAGCAA PCR-G-EMX(SEQ ID NO: 167) GAAAGGACGAAACACCGGAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAA PCR-G-VEG (SEQ ID NO: 168)GAAAGGACGAAACACC GGGGTGGGGGGAGTTTGCTCCGTTTTAGAGCTAGAAATAGCAAPrimers for Performing T7 Endonuclease I DNA Cleavage Assay.

(SEQ ID NO: 169) Survey_GFP-fwd TACGGCAAGCTGACCCTGAA (SEQ ID NO: 170)Survey_GFP-rev GTCCATGCCGAGAGTGATCC (SEQ ID NO: 171)Survey_CLTA-fwd GCCAGGGGCTGTTATCTTGG (SEQ ID NO: 172)Survey_CLTA-rev ATGCACAGAAGCACAGGTTGA (SEQ ID NO: 173)Survey_EMX-fwd CTGTGTCCTCTTCCTGCCCT (SEQ ID NO: 174)Survey_EMX-rev CTCTCCGAGGAGAAGGCCAA (SEQ ID NO: 175)Survey_VEGF-fwd CCACACAGCTTCCCGTTCTC (SEQ ID NO: 176)Survey_VEGF-rev GAGAGCCGTTCCCTCTTTGCPrimers for High-Throughput Sequencing of On-Target and Off-Target Sitesin Human Genome.

(SEQ ID NO: 177) HTS_EMX_ON-fwd CACTCTTTCCCTACACGACGCTCTTCCGATCTCCTCCCCATTGGCCTGCTTC (SEQ ID NO: 178) HTS_EMX_Off1-fwdCACTCTTTCCCTACACGACGCTCTTCCGATCT TCGTCCTGCTCTCACTTAGAC (SEQ ID NO: 179)HTS_EMX_Off2-fwd CACTCTTTCCCTACACGACGCTCTTCCGATCT TTTTGTGGCTTGGCCCCAGT(SEQ ID NO: 180) HTS_EMX_Off3-fwd CACTCTTTCCCTACACGACGCTCTTCCGATCTTGCAGTCTCATGACTTGGCCT (SEQ ID NO: 181) HTS_EMX_Off4-fwdCACTCTTTCCCTACACGACGCTCTTCCGATCT TTCTGAGGGCTGCTACCTGT (SEQ ID NO: 182)HTS_VEFG_ON-fwd CACTCTTTCCCTACACGACGCTCTTCCGATCT ACATGAAGCAACTCCAGTCCCA(SEQ ID NO: 183) HTS_VEGF_Off1- CACTCTTTCCCTACACGACGCTCTTCCGATCT fwdAGCAGACCCACTGAGTCAACTG (SEQ ID NO: 184) HTS_VEGF_Off2-CACTCTTTCCCTACACGACGCTCTTCCGATCT fwd CCCGCCACAGTCGTGTCAT(SEQ ID NO: 185) HTS_VEGF_Off3- CACTCTTTCCCTACACGACGCTCTTCCGATCT fwdCGCCCCGGTACAAGGTGA (SEQ ID NO: 186) HTS_VEGF_Off4-CACTCTTTCCCTACACGACGCTCTTCCGATCT fwd GTACCGTACATTGTAGGATGTTT(SEQ ID NO: 187) HTS_CLTA2_ON- CACTCTTTCCCTACACGACGCTCTTCCGATCT fwdCCTCATCTCCCTCAAGCAGGC (SEQ ID NO: 188) HTS_CLTA2_Off1-CACTCTTTCCCTACACGACGCTCTTCCGATCT fwd ATTCTGCTCTTGAGGTTATTTGT(SEQ ID NO: 189) HTS_CLTA2_Off2- CACTCTTTCCCTACACGACGCTCTTCCGATCT fwdCACCTCTGCCTCAAGAGCAGAAAA (SEQ ID NO: 190) HTS_CLTA2_Off3-CACTCTTTCCCTACACGACGCTCTTCCGATCT fwd TGTGTGTGTGTGTGTGTAGGACT(SEQ ID NO: 191) HTS_EMX_ON-rev GGAGTTCAGACGTGTGCTCTTCCGATCTTCATCTGTGCCCCTCCCTCC (SEQ ID NO: 192) HTS_EMX_Off-revGGAGTTCAGACGTGTGCTCTTCCGATCT CGAGAAGGAGGTGCAGGAG (SEQ ID NO: 193)HTS_EMX_Off-rev GGAGTTCAGACGTGTGCTCTTCCGATCT CGGGAGCTGTTCAGAGGCTG(SEQ ID NO: 194) HTS_EMX_Off-rev GGAGTTCAGACGTGTGCTCTTCCGATCTCTCACCTGGGCGAGAAAGGT (SEQ ID NO: 195) HTS_EMX_Off-revGGAGTTCAGACGTGTGCTCTTCCGATCT AAAACTCAAAGAAATGCCCAATCA (SEQ ID NO: 196)HTS_VEFG_ON-rev GGAGTTCAGACGTGTGCTCTTCCGATCT AGACGCTGCTCGCTCCATTC(SEQ ID NO: 197) HTS_VEGF_Off1- GGAGTTCAGACGTGTGCTCTTCCGATCT revACAGGCATGAATCACTGCACCT (SEQ ID NO: 198) HTS_VEGF_Off2-GGAGTTCAGACGTGTGCTCTTCCGATCT rev GCGGCAACTTCAGACAACCGA (SEQ ID NO: 199)HTS_VEGF_Off3- GGAGTTCAGACGTGTGCTCTTCCGATCT rev GACCCAGGGGCACCAGTT(SEQ ID NO: 200) HTS_VEGF_Off4- GGAGTTCAGACGTGTGCTCTTCCGATCT revCTGCCTTCATTGCTTAAAAGTGGAT (SEQ ID NO: 201) HTS_CLTA2_ON-GGAGTTCAGACGTGTGCTCTTCCGATCT rev ACAGTTGAAGGAAGGAAACATGC(SEQ ID NO: 202) HTS_CLTA2_Off1- GGAGTTCAGACGTGTGCTCTTCCGATCT revGCTGCATTTGCCCATTTCCA (SEQ ID NO: 203) HTS_CLTA2_Off2-GGAGTTCAGACGTGTGCTCTTCCGATCT rev GTTGGGGGAGGAGGAGCTTAT (SEQ ID NO: 204)HTS_CLTA2_Off3- GGAGTTCAGACGTGTGCTCTTCCGATCT revCTAAGAGCTATAAGGGCAAATGACT (SEQ ID NO: 205) HTS_EGFP-fwdCACTCTTTCCCTACACGACGCTCTTCCGA TCTNNNNACGTAAACGGCCACAAGTTC(SEQ ID NO: 206) HTS_EGFP-rev GGAGTTCAGACGTGTGCTCTTCCGATCTGTCGTCCTTGAAGAAGATGGTG (SEQ ID NO: 207) HTS_MusEMX_ON-CACTCTTTCCCTACACGACGCTCTTCCGATCT fwd CCAGGTGAAGGTGTGGTTCCAG(SEQ ID NO: 208) HTS_MusEMX_ON- GGAGTTCAGACGTGTGCTCTTCCGATCT revCCCCTAGTCATTGGAGGTGACResultsHighly Efficient Delivery of Cre Recombinase Fused to a SupernegativelyCharged Protein

It was speculated that imparting the highly anionic electrostaticproperties of nucleic acids to genome-editing proteins may enable theirefficient delivery into mammalian cells using cationic lipids (FIG.27(A)). For proteins of interest that are not natively highly negativelycharged, it was thought that fusion with a natural or engineeredsupernegatively charged protein¹⁷ would impart a polyanionic character.For nucleic acid-binding proteins, it was speculated that simplecomplexation with native DNA or RNA substrates might provide sufficientanionic character to support cationic lipid-based delivery (FIG. 27(A)).

It was first tested whether the engineered supernegatively charged GFPvariant,³⁵ (−30)GFP, could mediate encapsulation and delivery of fusedprotein cargo (FIG. 27(B)). (−30)GFP was fused to Cre recombinase andseveral commercially available cationic lipids were tested for theirability to functionally deliver the fusion into HeLa cells that onlyexpress DsRed upon Cre-mediated recombination (FIG. 28(A)). Delivery of10 nM (−30)GFP-Cre complexed with 1.5 μL Lipofectamine RNAiMAX(hereafter referred to as “RNAiMAX”, Life Technologies, Carlsbad Calif.)in media containing 10% fetal bovine serum (FBS) led to strong DsRedfluorescence signal among treated cells. Fluorescence-activated cellsorting (FACS) revealed that 48 hours after treatment 52% of cellsexpressed DsRed consistent with Cre recombination (FIG. 28(B)).

Optimization resulted in recombination efficiencies of 65% using 25 nM(−30)GFP-Cre complexed with 1.5 μL RNAiMAX in 250 μL of media containing10% FBS (FIG. 28(C)). The potency of lipid-mediated anionic Cre deliveryis notable compared to that of cationic protein-mediated delivery. Only1 nM (−30)GFP-Cre with cationic lipid was needed to result in 15-20%recombined cells, whereas 1 μM (+36)GFP-Cre was required to achieve thisextent of recombination, representing a 1,000-fold difference indelivery potency (FIG. 28(C)). Nearly identical results were observed ina second Cre reporter cell line (BSR TdTomato) (FIG. 33(A)). Increasingthe amount of cationic lipid increased toxicity (FIG. 33(B)) and it wasfound that 1.5 μL RNAiMAX per 250 μL sample maximized recombinationefficiency while inducing minimal cell toxicity. Under these conditions,cationic lipids did not increase the delivery potency of neutral orcationic Cre recombinase fusions (FIG. 28(C) and FIG. 33(C)), indicatingthat the strongly negative charge of (−30)GFP-Cre was required toparticipate in cationic lipid-mediated delivery. It was also observedthat increasing the amount of cationic lipid increased the concentrationof protein required for maximal recombination, consistent with a modelin which deliverable proteins are complexed with specificstiochiometries of cationic lipids (FIG. 28(D)). These observationscollectively indicate that cationic lipids can mediate the potentdelivery of polyanionic proteins into mammalian cells even in thepresence of serum.

To determine if the higher potency of cationic lipid-mediated(−30)GFP-Cre delivery relative to cationic protein-mediated deliveryarises from more total protein uptake by cells, or from a higherfraction of functional, non-endosomal protein molecules that entercells, flow cytometry was used to measure GFP fluorescence of cellstreated with either (+36)GFP-Cre or liposomal (−30)GFP-Cre under theirrespective optimal Cre delivery conditions. Comparison of cellularfluorescence and recombination efficiency reveals that lipid-mediatedfunctional delivery of (−30)GFP-Cre is 9,800-fold more potent per amountof endocytosed protein than delivery of (+36)GFP-Cre (FIG. 34). Takentogether, these results suggest that the unusually high potency oflipid-mediated delivery of anionic proteins does not arise fromunusually high protein uptake in each cell, but rather frompost-endocytosis processes that likely include endosomal escape into thecytoplasm and the avoidance of lysosomal protein degradation.

To test whether the ability to deliver polyanionic proteins is dependenton proprietary components in RNAiMAX or if other cationic lipids arecapable of mediating similarly potent delivery, several othertransfection reagents designed to deliver nucleic acids were tested(FIG. 28(E)). While RNAiMAX remained the most effective functionaldelivery agent for (−30)GFP-Cre, other cationic lipid formulations alsoresulted in potent delivery. Lipofectamine 2000 and Lipofectamine LTX(Life Technologies, Carlsbad Calif.), two plasmid transfection reagentsbased on cationic lipid formulations,²¹ and SAINT-Red (SynvoluxTherapeutics, Groningen Netherlands), an siRNA delivery formulationcontaining a synthetic pyridium-containing cationic lipid, all resultedin strong functional (−30)GFP-Cre delivery over a range ofconcentrations (FIG. 28(E)). In contrast, strong deliveries with thecationic lipid DOTAP (Roche Diagnostics, Indianapolis Ind.) or thepeptide-based nucleic acid delivery agent EZ-PLEX (Ascension Bio, TampaFla.) were not observed (FIG. 28(E)). These observations collectivelyindicate that several (but not all) cationic lipids are able toencapsulate and deliver negatively charged proteins into human cells.

It was speculated that it should be possible to use cationic lipids todeliver polyanionic proteins other than (−30)GFP. Engineered polyanionicprotein domains commonly used in biomedical research include the VP64activation domain (−22 net theoretical charge) widely used in fusionswith engineered zinc finger arrays, TALE repeat arrays, or dCas9 fortranscriptional activation, and 3×FLAG (−7 net theoretical charge), anepitope tag used for protein purification and visualization (FIG.28(F)). It was observed that both VP64 and 3× FLAG enhance functionaldelivery of Cre recombinase with cationic lipids, though not aseffectively as (−30)GFP, likely due to their lower overall negativecharge (FIG. 33(C)). These observations demonstrate that unusuallynegatively charged proteins beyond (−30)GFP can mediate efficientcationic lipid-based delivery into mammalian cells.

Functional Delivery of TALE Activator Proteins

The lipid-mediated delivery of TALE-VP64 transcriptional activators(approximately +4 theoretical net charge, depending on TALE variantused) into cultured human cells was tested. While modestly effectivecleavage of endogenous genes by delivered TALEN proteins has beendemonstrated in mammalian cells in the absence of serum using cationicpeptides such as Arg₉,³⁶ the delivery of TALE-based transcription factorproteins has not yet been reported, and no effective delivery of TALEproteins in serum has been previously described to our knowledge. Thegene for neurotrophin-3 (NTF3), a neural growth factor that has beenassociated with neurodegenerative diseases, was targeted.³⁷ A previouslydescribed NTF3-targeting TALE-VP64³⁸ was fused to (−30)GFP (FIG. 29(A))and treated HEK293T cells with 25 nM (−30)GFP-NTF3 TALE1-VP64 andRNAiMAX under the conditions optimized for Cre delivery. Gene expressionlevels of NTF3 4 hours after treatment were 3.5-fold higher in cellstreated with 25 nM (−30)GFP-NTF3 TALE-VP64 and RNAiMAX than untreatedcells, cells treated with RNAiMAX only, or cells treated with aVEGF-targeting TALE transcriptional activator (FIG. 29(B)). Comparablelevels of NTF3 expression were observed 48 hours after transfection ofplasmids encoding the same NTF3-targeting TALE-VP64 (FIG. 29(B)).

Since the synergistic expression of multiple TALE activators targetingdifferent sites on the same gene has been shown to augment geneactivation,³⁸ five distinct NTF3-targeting TALE activators fused to(−30) GFP using RNAiMAX were simultaneously delivered. Protein-lipidcomplexes were prepared as above by adding the five(−30)GFP-NTF3-TALE-VP64 proteins at 5 nM each, for a total of 25 nMprotein. A 6.5-fold increase in NTF3 expression was observed after a4-hour incubation (FIG. 29(B) and FIG. 35), while plasmidco-transfection of all five NTF3 TALE activators, followed by a 48-hourincubation, resulted in a 10-fold increase in NTF3 expression levels(FIG. 29(B)). These findings demonstrate that TALE activator proteinscan be delivered using cationic lipids to transiently activate geneexpression in human cells. The delivery of programmable transcriptionalactivator proteins may enable the one-time activation of a target genewhile avoiding chronic gene expression, a general concern with DNA-baseddelivery of programmable transcription factors. This capability mayprove especially valuable for proteins that effect a one-time permanentchange in cell state or cell fate when transiently expressed.³⁹

Highly Efficient Delivery of Cas9:sgRNA Protein:RNA Complexes into HumanCells

Given the potent lipid-mediated delivery of polyanionic Cre and TALEactivator protein variants in full-serum media, it was speculated thatCRISPR-Cas9:sgRNA complexes, either as fusions with (−30)GFP or asnative polyanionic Cas9:guide RNA complexes, might also be deliveredinto human cells using this approach. Using a well-establishedCas9-induced gene disruption assay,⁴⁰ specific sites within a genomicEGFP reporter gene in human U2OS cells were targeted (FIG. 36(A)).On-target Cas9 cleavage induces non-homologous end joining (NHEJ) inEGFP and the loss of cell fluorescence. To avoid interference from thefluorescence of (−30)GFP, a Y67S mutation was introduced into (−30)GFPto eliminate its fluorescence, and designated this non-fluorescentvariant as (−30)dGFP.

Treatment of U2OS reporter cells with 25 nM (−30)dGFP-NLS-Cas9 and 50 nMEGFP-targeting sgRNA with RNAiMAX in media containing 10% FBS showedloss of EGFP expression in 48% of cells (FIG. 30(A)). Cotransfection ofplasmids expressing Cas9 or sgRNA resulted in similar EGFP loss in 37%of cells (FIG. 30(A)). No significant EGFP disruption was observed upontransfection of plasmids encoding EGFP sgRNA alone, Cas9 alone, orcotransfection of plasmids encoding Cas9 and an sgRNA designed to targeta VEGF locus (FIG. 30(A), FIG. 36(B)). It was confirmed that the robustdisruption of EGFP was not a result of cellular toxicity (FIGS.36(C)-(D)). It was also observed that treatment of cells with(+36)dGFP-NLS-Cas9 and sgRNA in the presence of 10% FBS serum did notlead to efficient gene disruption (FIG. 30(A)), suggesting thatcationic-peptide based methods of delivery for Cas9 and sgRNA are noteffective perhaps due to interference of gRNA:Cas9 complex formation ornuclease function by superpositively charged proteins.⁴¹ Together, theseresults establish that cationic lipid-mediated delivery of(−30)dGFP-NLS-Cas9:sgRNA complexes can result in efficientsgRNA-dependent target gene disruption in human cells.

Polyanionic sgRNA is Necessary and Sufficient for EfficientLipid-Mediated Cas9 Delivery

Since the complex of native Cas9 protein (+22 net theoretical charge)and an sgRNA (˜103 anionic phosphate groups) should be overall highlyanionic, next it was tested if native Cas9:sgRNA complexes withoutfusion to polyanionic proteins can be delivered into human cells usingcationic lipids. Treatment of U2OS EGFP reporter cells with 100 nM Cas9,100 nM EGFP sgRNA, and 0.8 μL RNAiMAX resulted in 65% disruption of theEGFP reporter gene (FIG. 30(A)). Treatment of cells with Cas9 proteinand sgRNA, but without RNAiMAX, resulted in no loss of GFP fluorescence(FIG. 30(A)). These observations suggest that sgRNA alone, even in theabsence of a supernegatively charged fusion protein, can provide thehighly anionic character needed to mediate cationic lipid-based deliveryof Cas9.

Comparison of gene disruption efficiency arising from the cationiclipid-mediated delivery of (−30)dGFP-NLS-Cas9:sgRNA versus Cas9:sgRNArevealed that at low doses (−30)dGFP-NLS-Cas9 results in more efficientgene disruption than native Cas9 (FIG. 37(A)), it is outperformed bynative Cas9 at higher concentrations, as well as at the respectiveoptimal protein:sgRNA dose of either protein (FIGS. 37(B)-37(C)). Theseresults further establish that sgRNA can supply sufficient negativecharge to support cationic lipid-based delivery of complexed Cas9protein.

It was also observed that while overall less protein was required foroptimal delivery of (−30)dGFP-NLS-Cas9 than Cas9, a higher sgRNA:proteinratio was required for maximal (−30)dGFP-NLS-Cas9-mediated EGFP genedisruption than for native Cas9-mediated gene disruption (FIG. 37(D)).It was speculated that more equivalents of sgRNA are needed to complexwith (−30)dGFP-NLS-Cas9 since fused (−30)dGFP may electrostaticallyinterfere with Cas9:sgRNA complexation. As the ideal protein dose for(−30)dGFP-NLS-Cas9 mediated EGFP gene disruption is 10-fold lower thanthat of wild-type Cas9, the results also suggest that (−30)dGFP-Cas9 isbetter encapsulated by cationic liposomes than Cas9:sgRNA due to itshigher overall negative charge, but this charge magnitude may interferewith Cas9:sgRNA interactions, necessitating more sgRNA per protein andpotentially reducing total delivered Cas9 activity. In addition,NLS-Cas9 and Cas9-NLS proteins were generated and tested, and it wasobserved that while the presence of an NLS in (−30)dGFP-NLS-Cas9 couldat least partially explain differences in delivery efficacy at very lowconcentrations, Cas9, NLS-Cas9, and Cas9-NLS all result in higherefficiency of EGFP disruption than (−30)dGFP-NLS-Cas9 at 25 nM or higherconcentrations (FIGS. 38(A)-(C)).

Cas9:sgRNA delivery with cationic lipid formulations other than RNAiMAXwas also tested. Delivery with Lipofectamine 2000 was notably moreefficient than with RNAiMAX, resulting in up to 80% Cas9-mediated genedisruption (FIG. 39(A)), and maintaining high efficiency (60% genedisruption) even at 1 nM protein (FIG. 39(A)). However, due to thesomewhat higher toxicity of Lipofectamine 2000 compared to RNAiMAX undercell culture conditions (FIGS. 33(B)-C)), RNAiMAX was used for allsubsequent cell culture studies.

To verify that EGFP disruption arose from genome modification and notonly from Cas9 binding,⁴² the T7 endonuclease I (T7EI) assay⁴³ was usedto detect and quantify the frequency of Cas9-mediated genomicinsertion/deletion mutations (indels) at the target EGFP locus (FIG.30(B)). The T7EI assay results showed that only those cells treated withboth Cas9 and EGFP sgRNA plasmids, or Cas9 protein and purified EGFPsgRNA, contained indels at the target site. Taken together, thesefindings establish that active Cas9:sgRNA complexes can be potentlydelivered into human cells with cationic lipids in a manner dependent onthe negative charge provided by the sgRNA.

U2OS EGFP reporter cells were also treated with a single lipid-mediateddelivery treatment of Cas9 complexed with a mixture of four gRNAstargeting EGFP, CLTA, EMX, and VEGF. This treatment resulted inefficient disruption of all four targets, with cleavage efficiencies of58%, 28%, 16%, and 40%, respectively, as measured by T7E1 cleavageassay. These high gene disruption efficiencies from a single delivery of50 nM Cas9 and 25 nM of each sgRNA (100 nM total sgRNA) demonstrate thatlipid-mediated Cas9:sgRNA delivery can support efficient multiplexedgenome editing (FIG. 30(C)).

Functional Delivery of Cas9 Nickases and dCas9 Activators

Next, whether cationic lipid-based protein delivery could be extended todeliver other Cas9-derived genome engineering tools such as Cas9nickases⁴⁴ and Cas9-based transcriptional activators was tested.⁴⁵ Genedisruption efficiency in U2OS EGFP reporter cells resulting fromdelivery of Cas9 D10A nickase was measured, either by cotransfection ofnickase and appropriate paired EGFP-targeting sgRNA plasmids, or aspurified protein complexed with pairs of EGFP sgRNAs using RNAiMAX (FIG.30(D)). Both plasmid and cationic lipid-mediated protein:RNA delivery ofdual Cas9 nickases resulted in EGFP disruption with similar efficiencies(FIG. 30(D)) only in the presence of sgRNA pairs targeting oppositestrands, (sgRNA pairs g1+g5, and g3+g7), but not with sgRNA pairstargeting the same strand (sgRNA pair g5+g7) (FIG. 30(D)), consistentwith previous reports of Cas9 nickase cleavage requirements.⁴⁶

The NTF3 transcriptional activation efficiencies in HEK293T cellsresulting from either plasmid transfection or direct protein:sgRNAcomplex delivery of dCas9 fused to a VP64 activation domain were alsocompared.⁴⁵ Delivery of dCas9-VP64 activators either by plasmidtransfection or RNAiMAX-mediated protein delivery resulted in strong(≧˜10-fold) activation of NTF3 transcription (FIG. 30(E) and FIG. 40).Transcriptional activation levels resulting from plasmid transfectionwere more potent than activation resulting from protein delivery atoptimal assay times for each delivery method (FIG. 30(E)), potentiallydue to the sustained expression both Cas9 activator protein and sgRNAfrom the plasmids compared to the transient, single dose of purifiedprotein and RNA. While the above results indicate that such factors donot limit the potency of irreversible genome modification by deliveredCas9 nuclease and nickase proteins (FIGS. 40(A) and 40(D)), the low doseand transient nature of the delivered protein may more strongly limitpotency of dynamic processes such as transcriptional activation.Nevertheless, these results collectively indicate that both Cas9nickases and Cas9 transcriptional activators can also be deliveredeffectively by cationic lipid-mediated protein:RNA complex delivery.

Cas9:sgRNA delivery modifies genomes with greater specificity than DNAtransfection

DNA-free delivery of functional Cas9:sgRNA complexes circumvents risksassociated with viral or other gene delivery methods and has thepotential to improve the specificity of genome modification by avoidingthe unnecessary expression of genome-editing agent after the targetlocus is modified. To test if the described approach can disruptendogenous genes in human cells, genomic loci in the EMX1, CLTA2, andVEGF genes were targeted due to their potential biomedical relevance andtheir use in previous studies^(40,46,47) of Cas9 off-target cleavageactivity. Cationic lipid-mediated delivery of Cas9:sgRNA complexes intoHEK293T cells resulted in robust cleavage of all three human genes withefficiencies comparable to or greater than those of plasmid transfectionmethods as revealed by the T7EI assay using the same Cas9:sgRNA deliveryconditions previously optimized for U2OS cells (FIG. 31(A)).

To compare the endogenous gene modification specificity of plasmidversus protein:RNA delivery methods for Cas9, the on-target locus wasamplified as well as several known off-target sites (FIG. 44) fromgenomic DNA isolated from HEK293 cells treated either by transfection ofCas9 and sgRNA expression plasmids, or by RNAiMAX-mediated Cas9:sgRNAcomplex delivery under conditions that resulted in comparable on-targetmodification efficiencies. The indel frequencies at the three on-targetand 11 off-target sites were assayed by high-throughput DNA sequencing(FIG. 45). For all three target genes, the frequency of on-target DNAmodification resulting from either plasmid or protein:sgRNA delivery wasapproximately 10% (FIGS. 41(A)-(C)), enabling a comparison of off-targetmodification between the two techniques under treatment conditions thatresult in very similar on-target genome modification efficiencies.Importantly, the frequency of off-target genome modification for all 11off-target sites was lower from protein:sgRNA delivery compared withplasmid delivery, and as a result the ratio of on-target to off-targetmodification for all sites tested was up to 19-fold higher forprotein:sgRNA delivery than for plasmid delivery (FIGS. 31(B)-(D)).

DNA modification specificity was higher for protein:sgRNA delivery thanfor plasmid delivery at loci with high levels of off-target modification(such as the four VEGF off-target sites, for which plasmid deliveryyielded average on-target:off-target modification ratios between 4- and20-fold but protein:RNA delivery yielded average on-target:off-targetmodification ratios between 9- and 400-fold) as well as for loci withlower levels of off-target modification (such as the three EMXoff-target loci, for which plasmid delivery yielded averageon-target:off-target modification ratios as low as 64-fold butprotein:RNA delivery yielded average on-target:off-target modificationratios of 500- to 2,000-fold). Taken together, these results indicatethat the delivery of Cas9:sgRNA complexes using cationic lipids caneffect target gene modification at high efficiency and withsubstantially greater specificity than the delivery of DNA expressingCas9 and sgRNA.

Delivery of Cas9:sgRNA into Mouse Embryonic Stem Cells

The potent and transient cationic lipid-mediated delivery of Cas9:sgRNAto effect efficient, permanent, and highly specific gene editing couldbe especially useful in stem cells. To test this possibility, mouseembryonic stem cells expressing Tau-EGFP⁴⁸ were treated with Cas9 and anEGFP-targeting sgRNA. Under standard stem-cell culture conditions,EGFP-positive floating spheres were formed. The floating spheres weretreated with Cas9:sgRNA complexed with RNAiMAX, or with Cas9 and RNAiMAXwithout sgRNA as a control. Three days post-treatment, a reduction inGFP fluorescence in the Cas9:sgRNA-treated spheres compared to thecontrol samples was observed (FIG. 42(A)). The treated spheres weredissociated, and the cells were allowed to attach to a gelatin-coateddish and differentiate into progenitor cells. Immunohistochemistry usingan anti-GFP antibody confirmed knockdown of EGFP expression in the cellsof Cas9:sgRNA treated samples, with many nuclei lacking any apparentEGFP. In contrast, all cells derived from control spheres were EGFPpositive (FIG. 42(B)). Genomic DNA harvested from Cas9:sgRNA-treatedcells was subjected to T7EI assay, resulting in clear evidence of indelsat the Tau-EGFP locus (FIG. 42(C)). From this assay, an indel frequencyof 42% was calculated from both cationic lipid-mediated Cas9:sgRNAdelivery and transfection of Cas9 and sgRNA DNA. No target modificationwas detected in control samples lacking Cas9:sgRNA or containing Cas9and an unrelated gRNA. These findings demonstrate that cationiclipid-mediated Cas9:sgRNA delivery can effect highly efficient genedisruption in mouse embryonic stem cells.

In Vivo Cationic Lipid-Mediated Delivery of Cre Recombinase andCas9:sgRNA

The high-efficiency delivery of functional genome-editing proteins invivo enables a wide range of applications including non-viraltherapeutic genome editing to correct genetic diseases. To evaluate theprotein delivery method described above in a living mammal, delivery tothe mouse inner ear was chosen, due to its confined space,well-characterized inner ear cell types, and the existence of geneticdeafness mouse models that may enable future hearing recovery studies.The in vivo deliveries of two types of proteins into the mouse inneryear were attempted. First, the delivery of (−30)GFP-Cre protein wastested to assess the targeting of inner ear cell types and theefficiency of functional protein delivery. Second, the delivery ofCas9:sgRNA complexes to the inner ear were evaluated to determine ifcationic lipid-mediated protein:gRNA complex delivery can supportCRISPR-based gene editing in vivo.

It has been previously shown that (+36)GFP-Cre can be delivered to mouseretina,¹⁶ although the protein resulted in only modest levels ofrecombinant conversion suggestive of inefficient in vivo delivery. Forour initial inner ear delivery trials, (−30)GFP-Cre was complexed withRNAiMAX and the complex was injected into the cochlea of postnatal day 0(P0) reporter mice with a genomically integrated floxed-STOP tdTomatoreporter. As with the previously described in vitro Cre reporter cellline, functional delivery of Cre to the inner ear cells, followed byendosomal escape, nuclear localization, and Cre-mediated recombinationresults in expression of tdTomato. After injection, the cochleas wereharvested for immunolabeling with inner ear cell markers forco-localization with tdTomato. RNAiMAX injection alone was used ascontrol. Five days following injection of (−30)GFP-Cre and RNAiMAX,cochlear outer hair cells, the auditory sensory cells that detect sound,showed strong tdTomato signal that co-localized with the hair cellmarker myosin VIIa (Myo7a), demonstrating functional Cre delivery tohair cells (FIGS. 32(A)-(B)). No tdTomato expression was detected incontrol cochleas (FIG. 32(A)). The tdTomato signal was concentrated inthe region of the injection site at the basal turn of the cochlea. Onaverage 33±3% of outer hair cells were tdTomato positive at the base ofthe cochlea (P<0.001; mean±SEM, n=4).

To further determine the effect of cationic lipid-mediated (−30)GFP-Creprotein delivery on targeted cells, hair cell stereocilia, a delicatestructure that is essential for hearing, were examined 10 dayspost-injection. TdTomato positive outer hair cells had typicalstereocilia structure as imaged by espin expression, similar to controlstereocilia (FIG. 32(B)). No tdTomato expression was detected in controlcochleas. These observations indicate that cationic lipid-mediateddelivery of (−30)GFP-Cre protein effects recombination in cochlear outerhair cells without apparently affecting hair cell architecture.

Because target volume, protein dose, and sgRNA dose in vivo aredifferent than in cell culture experiments, the above experiments wererepeated under different delivery conditions. Delivery usingLipofectamine 2000 was tested due to its higher potency in vitro (FIG.39(A)) and dramatically higher recombination efficiency was observed:over 90% outer hair cells in cochleas treated with(−30)GFP-Cre+Lipofectamine 2000 were tdTomato positive (FIG. 32(C)). Incomparison to control samples, some outer hair cell loss was observed(FIG. 32(C)), consistent with the previous observation of the highercell toxicity of Lipofectamine 2000, although the overall cochleararchitecture was preserved.

To test the effectiveness of Cas9:sgRNA delivery in vivo, Cas9 and sgRNAtargeting EGFP were combined with RNAiMAX and the resulting complexeswere injected into postnatal day 2 (P2) transgenic Atoh1-GFP mousecochlea in which all hair cells express GFP under the control of a haircell-specific enhancer for transcription factor Atoh1.⁴⁹ Using thismodel, Cas9:sgRNA-mediated disruption of EGFP results in loss of EGFPfluorescence in outer hair cells. Ten days after injection of Cas9:sgRNAwith cationic lipid, the absence of GFP was observed in 13% of outerhair cells near the injection site. In contrast, control cochleainjected with Cas9 protein and RNAiMAX without any sgRNA showed no lossof EGFP signal (FIG. 32(D)). The outer hair cells of cochlea injectedwith Cas9:sgRNA RNAiMAX complexes appeared to be otherwise unaffected,with stereotypical expression of Myo7a and healthy nuclei, consistentwith minimal hair cell toxicity (FIG. 32(D)). High-throughput DNAsequencing of genomic DNA isolated from cochlea tissue samples revealedindels consistent with GFP target gene disruption in the treatedsamples, but not in the control samples that lacked sgRNA (FIG. 43(A)).In addition, the inner ear in vivo delivery of Cas9:sgRNA using an sgRNAthat targets the EMX gene was repeated and indels in the EMX gene intreated animals, but not control animals were similarly observed (FIG.43(B))

As (−30)GFP-Cre complexed with Lipofectamine 2000 resulted in moreefficient modification of the target hair cell population than(−30)GFP-Cre complexed with RNAiMAX (FIGS. 32(A) and 32(C)), its use onCas9:sgRNA delivery to Atoh1-GFP cochlea was tested as above. Loss ofGFP expression was observed in 20% of outer hair cells near theinjection site after 10 days, whereas all outer hair cells maintainedstrong GFP expression in control cochlea injected with Cas9 andLipofectamine 2000 but no sgRNA (FIG. 32(D)). In contrast to modest haircell loss observed following Lipofectamine 2000 delivery of (−30)GFP-Cre(FIG. 32(C)), outer hair cells targeted by Cas9:sgRNA exhibited noobvious toxicity or structural alteration (FIG. 32(D)).

As with (−30)GFP-Cre, virus-free, cationic lipid-mediated delivery ofCas9:sgRNA into the mouse inner ear successfully modified a specificgenomic locus in the outer hair cell population, leading to loss oftarget gene expression. Nearly half of all types of genetic deafnessarise from hair cell loss or dysfunction,⁵⁰ the results presented hereinsuggest a potential strategy based on the delivery of Cas9:sgRNAcomplexes to genetically modify these cells to effect hearing recovery.

Determination of Protein Delivery Efficacy for (−30)GFP-Cre

To determine if the higher potency of liposome-mediated (−30)GFP-Credelivery compared with that of cationic protein delivery arises frommore total protein uptake by cells or from a higher fraction offunctional, non-endosomal protein molecules taken up by the cells, flowcytometry was used to measure GFP fluorescence of cells treated witheither (+36)GFP-Cre or liposomal (−30)GFP-Cre under their respectiveoptimal Cre delivery conditions. Cell fluorescence reports totalendocytosed (−30)GFP-Cre or (+36)GFP-Cre regardless of endosomal ornon-endosomal localization.¹ Lipid-mediated protein delivery resulted insurprisingly small increases in total protein uptake (FIG. 34(A)),despite the high efficiency of lipid-mediated functional Cre delivery.While (+36)GFP-Cre treatment increased cellular GFP fluorescence by upto three orders of magnitude in a dose-dependent manner (FIG. 34(A)),consistent with previous reports,^(1,2) liposomal (−30)GFP-Cre treatmentinduced at most 5-fold increases in cellular GFP fluorescence (FIG.34(A)). Comparison of cellular fluorescence and recombination efficiencyreveals that lipid-mediated functional delivery of (−30)GFP-Cre is9,800-fold more potent per amount of endocytosed protein than deliveryof (+36)GFP-Cre (FIG. 34(B)).

To test if complexation of anionic (−30)GFP with cationic lipidsinterferes with GFP fluorescence and thus masks the true amount of cargothat enters the cell mCherry, which is fluorescent but not highlyanionic, was fused to either (−30)GFP or (+36)GFP and delivered bothprotein fusions to HeLa cells. After washing away protein that may haveadhered to cell surface but did not enter the cell with PBS+heparin (20U/mL), the cells were analyzed by FACS for mCherry fluorescence 4 hoursand 24 hours after treatment. It was observed that lipid-mediateddelivery of (−30)GFP-fused mCherry results in only slight increases incellular mCherry fluorescence, whereas mCherry fluorescence upondelivery of (+36)GFP-mCherry was generally ≧100-fold higher (FIG. 34(C))suggesting that fusion to (−30)GFP does not cause substantial amounts ofprotein cargo to enter the cell. Moreover, addition of lipids to(−30)GFP-Cre did not measurably alter the GFP fluorescence signal (FIG.34(D)), despite the fact that cationic lipids and anionic (−30)GFPclearly interact. Taken together, these results suggest that theunusually high potency of lipid-mediated delivery of anionic proteinsdoes not arise from unusually high protein uptake in each cell, butrather from post-endocytosis processes that likely include avoidance ofprotein degradation and endosomal escape into the cytoplasm.

Sensitivity Limit of Off-Target Cleavage Assays

The sensitivity of the high-throughput sequencing method for detectinggenomic off-target cleavage is limited by the amount genomic DNA (gDNA)input into the PCR amplification of each genomic target site. A 1 ngsample of human gDNA represents only approximately 330 unique genomes,and thus only approximately 330 unique copies of each genomic site arepresent. PCR amplification for each genomic target was performed on atotal of 150 ng of input gDNA, which provides amplicons derived from atmost 50,000, unique gDNA copies, respectively. Therefore, thehigh-throughput sequencing assay cannot detect rare genome modificationevents that occur at a frequency of less than 1 in 50,000 (0.002%). Thislimit is noted in Table 2.

Taken together, these findings suggest that cationic lipid-mediateddelivery of genome-editing proteins can serve as a powerful tool and anin vivo strategy for the treatment of genetic disease.

Conclusions

Efficient intracellular protein delivery in vitro and especially in vivohas been a persistent challenge in biomedical research and proteintherapeutics. While delivery using cationic peptides and proteins hasbeen widely studied for over two decades, sensitivity to serum proteins,neutralization by antibodies, degradation by extracellular andintracellular proteases, and poor endosomal escape post-internalizationhave limited the scope of protein delivery applications using thatapproach.

In the current Example, a general strategy for protein delivery thatmakes use of anionic protein complexation with cationic liposomes isdemonstrated. This method was used to deliver diverse protein classes,including the Cre tyrosine recombinase, TALE transcriptional activators,and Cas9 nucleases, nickases, and transcriptional activators (FIG.27(A)) to cultured cell lines, stem cell colonies, and therapeuticallyrelevant in vivo sites within the mouse inner ear. The describedapproach is highly efficient, producing modification rates on par withestablished nucleic acid transfection methods in cell culture, andenabling Cre recombinase and Cas9-mediated genome modification rates ofup to 90% and 20%, respectively, within the inner ear hair cellpopulation of live mice (FIGS. 32(C)-(D)). These results also suggestthat it may be possible to use cationic lipids to efficiently deliverother nucleic acid-binding proteins, including transcription factorsthat induce therapeutically relevant changes in cell fate, by complexingthem with nucleic acids.

Cationic lipid-based anionic protein delivery outperforms a potentcationic protein delivery fusion partner, (+36)GFP, by up to 9,800-foldper amount of endocytosed protein, inducing more efficient modificationof treated cells with orders of magnitude lower doses of protein (FIG.28(C) 34). For Cas9 nuclease delivery, this approach also resultsin >10-fold more specific genome modification than traditional plasmidtransfection (FIGS. 31(B)-(D)), likely due to the transient window ofCas9 activity to which each genome is exposed compared to DNA deliverymethods, consistent with previous reports.⁵¹

The described approach is simple to implement, requiring only thepurified deliverable protein and the use of popular commercial nucleicacid transfection reagents (FIG. 27(B)). Rendering a given proteinamenable to this approach requires simple translational fusion to ahighly anionic partner, such as (−30)GFP (FIG. 27(A)), and is eveneffective with common translational fusion tags including the VP64activation domain, and the 3×FLAG affinity tag (FIG. 28(F) and FIG.33(C)). In certain cases, as with the Cas9 protein, pre-complexationwith a cognate nucleic acid (sgRNA in this case) is sufficient (FIG.30(A)), as the partially exposed bound nucleic acid likely providessufficient anionic charge to mediate complexation with cationic lipids.

Others groups have reported the in vivo delivery of Cas9 expressionconstructs in DNA or mRNA form.^(52,53) The present Example demonstratesthat protein delivery is a viable approach to in vivo genome editing.

TABLE 1 EMX_On GAGTCCGAGCAGAAGAAGAAGGG (SEQ ID NO: 209) EMX_Off1GAGgCCGAGCAGAAGAAagACGG (SEQ ID NO: 210) EMX_Off2GAGTCCtAGCAGgAGAAGAAGaG (SEQ ID NO: 211) EMX_Off3GAGTCtaAGCAGAAGAAGAAGaG (SEQ ID NO: 212) EMX_Off4GAGTtaGAGCAGAAGAAGAAAGG (SEQ ID NO: 213) VEGF_On GGGTGGGGGGAGTTTGCTCCTGG(SEQ ID NO: 214) VEGF_Off1 GGaTGGaGGGAGTTTGCTCCTGG (SEQ ID NO: 215)VEGF_Off2 GGGaGGGtGGAGTTTGCTCCTGG (SEQ ID NO: 216) VEGF_Off3cGGgGGaGGGAGTTTGCTCCTGG (SEQ ID NO: 217) VEGF_Off4GGGgaGGGGaAGTTTGCTCCTGG (SEQ ID NO: 218) CLTA_On GCAGATGTAGTGTTTCCACAGGG(SEQ ID NO: 219) CLTA_Off1 aCAaATGTAGTaTTTCCACAGGG (SEQ ID NO: 220)CLTA_Off2 cCAGATGTAGTaTTcCCACAGGG (SEQ ID NO: 221) CLTA_Off3ctAGATGaAGTGcTTCCACATGG (SEQ ID NO: 222)Table 1. On-target and known off-target substrates of Cas9:sgRNAs thattarget sites in EMX, VEGF, and CLTA. A list of genomic on-target andoff-targets sites of the EMX, VEGF, and CLTA are shown with mutationsfrom the on-target sequence shown in lower case and bold. PAMs are shownin underline.

TABLE 2 Mock Plasmid Protein: sgRNA CLTA Sites treatment transfectiondelivery CLTA_On Indels 14 1228 1498 Total 10000 10000 10000 Modified(%) 0.140 12.280 14.980 P-value <1.0E−300 <1.0E−300 On: off specificity1 1 1 CLTA_Off 1 Indels 7 29 14 Total 41518 205204 125370 Modified (%)0.017 0.014 0.011 P-value 6.6E−01 4.5E−01 On: off specificity 869 1341CLTA_Off2 Indels 5 11 8 Total 25338 83944 54409 Modified (%) 0.020 0.0130.015 P-value 5.5E−01 5.7E−01 On: off specificity 937 1019 CLTA_Off3Indels 6 22 8 Total 41643 189886 76863 Modified (%) 0.014 0.012 0.010P-value 6.2E−01 5.8E−01 On: off specificity 1060 1439 Mock PlasmidProtein: sgRNA EMX Sites treatment transfection delivery EMX_On Indels 3930 1140 Total 10000 10000 10000 Modified (%) 0.030 9.300 P-value1.6E−264 <1.0E−300 On: off specificity 1 1 1 EMX_Off 1 Indels 0 6 6Total 24623 90935 100778 Modified (%) <0.002 0.007 P-value 3.5E−016.1E−01 On: off specificity 1409 1915 EMX_Off2 Indels 16 53 38 Total36061 204068 130084 Modified (%) 0.044 0.026 P-value 6.4E−02 1.8E−01 On:off specificity 358 390 EMX_Off3 Indels 20 147 44 Total 32575 157848110878 Modified (%) 0.061 0.093 P-value 8.1E−02 1.3E−01 On: offspecificity 100 287 EMX_Off4 Indels 16 141 23 Total 45548 86586 73451Modified (%) 0.035 0.163 P-value 2.8E−12 7.4E−01 On: off specificity 57364 Mock Plasmid Protein: sgRNA VEGF Sites treatment transfectiondelivery VEGF_On Indels 1 989 785 Total 10000 10000 Modified (%) 0.0109.890 7.850 P-value 1.5E−285 5.7E−228 On: off specificity 1 1 1VEGF_Off1 Indels 4 4240 602 Total 38625 184554 Modified (%) 0.010 2.2970.394 P-value <1.0E−300 3.7E−52 On: off specificity 4 20 VEGF_Off2Indels 5 727 18 Total 30301 79164 Modified (%) 0.017 0.918 <0.002P-value 4.7E−93 1.3E−04 On: off specificity 11 3925 VEGF_Off3 Indels 2536 21 Total 26379 110902 Modified (%) 0.008 0.483 0.022 P-value 2.0E−462.0E−01 On: off specificity 20 352 VEGF_Off4 Indels 0 1531 45 Total26012 122403Table 2. Indel frequencies, P values, and on-target:off-target cleavagespecificity ratios for EMX, CLTA, and VEGF on-target sites and 11 knownoff-target sites. CLTA sites: Total: total number of sequence counts;only the first 10,000 sequences were analyzed for the on-target sitesequences. Modified: number of indels divided by total number ofsequences as percentages. Upper limits of potential modification werecalculated for sites with no observed indels by assuming there is lessthan one indel then dividing by the total sequence count to arrive at anupper limit modification percentage, or taking the theoretical limit ofdetection (1/49,500; see Results above), whichever value was larger.P-values: for mock treatment, Cas9 plasmid transfection, and liposomalCas9 protein:sgRNA delivery, P-values were calculated as using atwo-sided Fisher's exact test between each CLTA-targeted treatmentsample (either DNA transfection or protein:sgRNA delivery) versus thecontrol sample (mock treatment) treated with Cas9 protein and an sgRNAtargeting EGFP. On:off specificity is the ratio of on-target tooff-target genomic modification frequency for each site. EMX sites showsthe experimental and analytic methods of CLTA analysis applied to EMXtarget sites. VEGF sites shows the experimental and analytic methods ofCLTA analysis as applied to VEGF target sites. Indel numbers in the mocktreatment control were subtracted from both plasmid transfection andprotein:sgRNA delivery indel numbers for determining total number ofindels and for calculating on-target:off-target ratios in FIG. 31 in themain text and also for FIG. 41.

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EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments, described herein. The scope of the present invention is notintended to be limited to the above Description, but rather is as setforth in the appended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for any of the purposes disclosed herein are included, andmethods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is also noted that theterm “comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anysupercharged protein; any nucleic acid; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

What is claimed is:
 1. A composition comprising a Cas9 protein and acationic lipid, wherein the Cas9 protein is associated with a guide RNA(gRNA), and the composition is capable of delivering the Cas9 proteinassociated with the gRNA to the nucleus of a cell, wherein the Cas9protein comprises an amino acid sequence that is at least 90% identicalto the amino acid sequence of SEQ ID NO: 44, or the Cas9 protein is anuclease-inactivated Cas9 (dCas9) that comprises an amino acid sequencethat is at least 90% identical to the amino acid sequence of SEQ ID NO:45 and comprises a D10A and/or a 11840A substitution as compared to theamino acid sequence of wild type Cas9; wherein the Cas9 protein isassociated with a supercharged protein having a net negative charge ofat least −5 at pH 7.4, and exhibiting a charge:molecular weight ratio ofgreater than 0.8, wherein the association has a net negative charge atpH 7.4; and the cationic lipid is Lipofectamine® 2000 or Lipofectamine®RNAiMAX.
 2. The composition of claim 1, wherein the composition exhibitslow toxicity when administered to a population of cells.
 3. Thecomposition of claim 2, wherein at least 60% of the cells are viablefollowing administration of the composition.
 4. The composition of claim1, wherein the dCas9 protein is fused to a transcriptional activator ora transcriptional repressor.
 5. The composition of claim 4, wherein thetranscriptional activator is selected from the group consisting of VP16,VP64, and p65.
 6. The composition of claim 1, wherein the dCas9 proteinis fused to a nuclease domain.
 7. The composition of claim 6, whereinthe nuclease domain comprises a FokI nuclease domain.
 8. The compositionof claim 1, wherein the dCas9 protein is fused to a recombinasecatalytic domain.
 9. The composition of claim 8, wherein the recombinasecatalytic domain comprises a Hin recombinase catalytic domain, a Ginrecombinase catalytic domain, or a Tn3 recombinase catalytic domain. 10.The composition of claim 1, wherein the dCas9 protein is fused to adeaminase.
 11. The composition of claim 10, wherein the deaminasecomprises a cytidine deaminase selected from the group consisting ofapolipoprotein B mRNA-editing complex 1 (APOBEC1), activation-inducedcytidine deaminase (AID), and APOBEC1 complementation factor/APOBEC1stimulating factor (ACF1/ASF) deaminases.
 12. The composition of claim1, wherein the dCas9 protein is fused to an epigenetic modifier.
 13. Thecomposition of claim 12, wherein the epigenetic modifier is selectedfrom the group consisting of histone demethylases, histonemethyltransferases, hydroxylases, histone deacetylases, and histoneacetyltransferases.
 14. The composition of claim 13, wherein theepigenetic modifier comprises the lysine (K)-specific demethylase 1A(LSD1) histone demethylase or ten-eleven translocation 1 (TET1)hydroxylase.
 15. The composition of claim 1, wherein the superchargedprotein is a fluorescent protein.
 16. The composition of claim 1,wherein the composition is a pharmaceutical composition.
 17. A methodcomprising administering the pharmaceutical composition of claim 16, toa subject in need thereof.
 18. A method of introducing a Cas9 proteininto a cell, the method comprising contacting the cell with thecomposition of claim 1, under conditions suitable for the Cas9 proteinto enter the cell, thereby introducing the Cas9 protein into the cell.19. The composition of claim 5, wherein the transcriptional repressor isa Kruppel associated box (KRAB) protein or a mSin3 interaction domain(SID) protein.
 20. The composition of claim 10, wherein the deaminasecomprises an adenosine deaminase.
 21. The composition of claim 20,wherein the adenosine deaminase comprises an adenosine deaminase actingon tRNA (ADAT) family deaminase.
 22. The composition of claim 1, whereinthe association is a covalent association.
 23. The composition of claim15, wherein the supercharged fluorescent protein is a supernegativelycharged green fluorescent protein (GFP).
 24. The composition of claim 1,wherein the Cas9 protein comprises an ortholog of Streptococcus pyogenesCas9.
 25. The composition of claim 10, wherein the deaminase comprises acytidine deaminase selected from the group consisting of APOBEC1 andAID.